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Transcript of Chapter 8: Expression and Modification of Recombinant Proteins I. Prokaryotic expression system II....
Chapter 8 Chapter 8 Expression and Expression and Modification of Recombinant Modification of Recombinant
ProteinsProteins
I Prokaryotic expression system
II Eukaryotic expression system1 Yeast expression2 Insect expression system3 Mammalian expression system
ReferencesReferences
1 Chapter 11 and 13 (Essentials of Molecular Biology)
2 Chapter 10 (An Introduction to Genetic Engineering)
I Prokaryotic I Prokaryotic Expression SystemExpression System
The red boxes represent exons the blue boxes represent the introns and the grey boxes represent the 5rsquo and 3rsquo UTRs
Insert only the ORF into an expression vector
that contains prokaryotic transcriptional and translational regulatory sequences
How do you express this gene in How do you express this gene in bacteriabacteria
Six Step ProcessSix Step Process
① Isolation of gene of interest② Introduction of gene to expression
vector③ Transformation into host cells④ Growth of cells through fermentation⑤ Isolation amp purification of protein⑥ Formulation of protein product
Cloning ProcessCloning Process Gene of interest is cut
out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same REs
Gene is inserted into plasmid and ligated with ligase
New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
ReferencesReferences
1 Chapter 11 and 13 (Essentials of Molecular Biology)
2 Chapter 10 (An Introduction to Genetic Engineering)
I Prokaryotic I Prokaryotic Expression SystemExpression System
The red boxes represent exons the blue boxes represent the introns and the grey boxes represent the 5rsquo and 3rsquo UTRs
Insert only the ORF into an expression vector
that contains prokaryotic transcriptional and translational regulatory sequences
How do you express this gene in How do you express this gene in bacteriabacteria
Six Step ProcessSix Step Process
① Isolation of gene of interest② Introduction of gene to expression
vector③ Transformation into host cells④ Growth of cells through fermentation⑤ Isolation amp purification of protein⑥ Formulation of protein product
Cloning ProcessCloning Process Gene of interest is cut
out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same REs
Gene is inserted into plasmid and ligated with ligase
New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
I Prokaryotic I Prokaryotic Expression SystemExpression System
The red boxes represent exons the blue boxes represent the introns and the grey boxes represent the 5rsquo and 3rsquo UTRs
Insert only the ORF into an expression vector
that contains prokaryotic transcriptional and translational regulatory sequences
How do you express this gene in How do you express this gene in bacteriabacteria
Six Step ProcessSix Step Process
① Isolation of gene of interest② Introduction of gene to expression
vector③ Transformation into host cells④ Growth of cells through fermentation⑤ Isolation amp purification of protein⑥ Formulation of protein product
Cloning ProcessCloning Process Gene of interest is cut
out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same REs
Gene is inserted into plasmid and ligated with ligase
New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
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- Slide 7
- Slide 8
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- Slide 36
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Six Step ProcessSix Step Process
① Isolation of gene of interest② Introduction of gene to expression
vector③ Transformation into host cells④ Growth of cells through fermentation⑤ Isolation amp purification of protein⑥ Formulation of protein product
Cloning ProcessCloning Process Gene of interest is cut
out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same REs
Gene is inserted into plasmid and ligated with ligase
New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
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- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Cloning ProcessCloning Process Gene of interest is cut
out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same REs
Gene is inserted into plasmid and ligated with ligase
New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Fundamentals of Gene Fundamentals of Gene ExpressionExpression
① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
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- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy
② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts
④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Prokaryotic ExpressionProkaryotic ExpressionPros
ConvenientEasy Produce and purify protein using the least expensi
ve and easiest reagents and equipment Best for large scale production of protein
Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need
ed for specific activity
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Which VectorWhich VectorPromoters
arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签
His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
T7 Promoter
lac Operator
RBS ATG TAG
peptide tag V5 poly-(his)
peptide tag V5 poly-(his)
mutiple cloning site (mcs)
Gene ( with without stop codon )
Fusion ProteinFusion Protein
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
A Generic Vector
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
II Eukaryotic Expression II Eukaryotic Expression SystemSystem
1 Yeast expression
2 Insect expression system
3 Mammalian expression system
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei
ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo
rt of the recombinant gene product Usually safe to use
Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu
karyote proteins
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Yeast Expression Vector
Pichia pastoris
Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
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- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Yeast are single celled eukaryotes Behave like bacteria but have key advan
tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)
Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
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- Slide 14
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- Slide 23
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- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Cloning in Yeast Cells① Uses a special plasmid that works both in E
coli and Yeast② Once gene of interest is inserted into this pl
asmid it must be linearized (cut open so it isnrsquot circular)
③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be
④ Now gene of interest is under control of the powerful AOX promoter
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
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- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
CloningCloning
12
3
4
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
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- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
2 Insect Expression2 Insect ExpressionPros
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function assays
Cons Expensive Sometimes proteins are not correctly folded Often not stable
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process
5rsquo 3rsquo
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5rsquo 3rsquoCloned gene
RecombinantAcMNPV DNA
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Baculovirus
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
BacVirus Expression System
pIExtradeInsect Cell Expression Plasmids (Novagen)
Bac-N-BluetradeBaculovirus Expression System (Invitrogen)
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
3 Mammalian Expression3 Mammalian Expression Pros
Produce protein in the most native and active form Have required PTM machinery to produce active
and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant
proteins Limitations on the mechanisms of protein
expression induction Almost always have over expression
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
HeLa cells in culture
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
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- Slide 24
- Slide 25
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- Slide 30
- Slide 31
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- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation
HeLa cells from the Nikon microsc
ope web site
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
HeLa Human cells
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
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- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection
HeLa cells as you will see them
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
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- Slide 19
- Slide 20
- Slide 21
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- Slide 24
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- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Expression System Expression System SelectionSelection
Choice depends on size and character of protein
Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture
High yields low cost Choose E coli Post-translational modifications essential Choo
se yeast baculovirus or other eukaryote
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Which VectorWhich Vector Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)
Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Key Parts to a VectorKey Parts to a Vector
Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid
Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive
Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene
Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
- Slide 12
- Slide 13
- Slide 14
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- Slide 21
- Slide 22
- Slide 23
- Slide 24
- Slide 25
- Slide 26
- Slide 27
- Slide 28
- Slide 29
- Slide 30
- Slide 31
- Slide 32
- Slide 33
- Slide 34
- Slide 35
- Slide 36
-
Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated
in bacterial cells Mammalian cells transformed by electroporati
on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
- Slide 1
- Slide 2
- Slide 3
- Slide 4
- Slide 5
- Slide 6
- Slide 7
- Slide 8
- Slide 9
- Slide 10
- Slide 11
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Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in005 uM Mtx
Culture aColony of cells
Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
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Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion
Grow in50 uM Mtx
Grow in025 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
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Mammalian ExpressionSystems
Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)
Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
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Mammalian Cell-line Mammalian Cell-line ExpressionExpression
Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
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SummarySummary1 The mechanism of regulation of gene expres
sion in prokaryotes is greatly different from eukaryotes
2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation
3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development
4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose
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- Slide 2
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