Overview of Gene Expression Systems with Gateway® Technology
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Research trends
• Current research focuses on proteomics: drug target example
– Approximately 35,000 human genes in the genome
– Estimated 500,000 targets for drug action
– The correlation between gene expression and protein expression is less than 10%
– Proteins need to be characterized in order to identify drug targets…need to express to characterize
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• Structural Proteomics - Protein Production– Make lots of protein to use in other experiments
• Interactions• Structure
– Make lots of protein to be used as a therapeutic (bioproduction)
• Functional Proteomics - Protein Expression – Study effects of protein expression in a cell – Identify the cellular functions of a protein– Over expression or gene knockdown– Study the function of a protein in different types of cells
Gene expression areas of study
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The Central Dogma
DNA RNA Protein
5
Buy or Isolate Gene
Determine Expression System
Express Protein in Culture
Analyze the Recombinant Protein
Transfer gene into Expression Vector
Select gene and enter into chosen system
Generate recombinant protein and analyze
The key steps in gene expression
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Generating Recombinant Protein - Overview
Buy or Isolate Gene
Determine Expression System
Express Protein in Culture
Analyze the Recombinant Protein
Transfer gene into Expression Vector
See Getting into
Gateway® slides
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Generating Recombinant Protein - Overview
Buy or Isolate Gene
Determine Expression System
Express Protein in Culture
Analyze the Recombinant Protein
Transfer gene into Expression Vector
Select gene and enter into chosen system
Generate recombinant protein and analyze
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Five primary systems used for expression…
Mammalian
Insect
Yeast
E. coli
in vitro
(aka cell free)
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Bacteria Yeast Insect Mammalian
Time Requirement
Cost of mediaand equipment
PTM / Probability of protein function
Ease of Use
Protein Production
Functional Analysis
In vitro
Choosing an expression system
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Protein production-getting enough protein
• Quantity (How much protein do you require?)
ng
μg
mg
g
kg
MammalianInsectYeastBacterial in vitro
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Protein production - post-translational modifications?
Are PTMs required?
No/Don’t Know
In vitro (Expressway™
Plus)
Bacterial (pET vectors)
Pull down InteractionStudiesToxic(µgs)
StructuralStudies
(mg to g)
Structural studies; antigen production
(mg to g)
pull down studies
Insect (Baculodirect™)
Mammalian(FreeStyle™)
Yes
-What kind of experiments are you doing?-How much protein do you need?
-What kind of experiments are you doing?-How much protein do you need?
Express in Eukaryotic System
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Protein production - typical challenges
• Solubility (Do you have difficulty expressing your protein in bacteria?)
Use fusions to improve solubility Try a eukaryotic system
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Gene
Gene
Gene
Gene
Gene
Gene
GeneGene
In Vitro
Tags
Your Vector
Viral System
Mammalian
Baculovirus
Yeast
E. coli Entry Clone
Gene
Get into any expression system with Gateway® Technology
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Gene Expression in E. coli
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Optimization of Protein Expression
-Gal GFP -Gal
- + - + - + - + - + - +
6xHis Fusion2
6xHis-TrxFusion
E. coli strain BL21 SI (salt-inducible, T7 promoter)
-GalGUS GFP
- + - + - + - + - + - +
GST Fusion1
1 pDEST™15 2 pDEST™17
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Expression Vector Design
B2B1
Apr
Gene
ATG Stop
Promoter
rbs
Native Proteins
B2B1
Apr
Gene
ATG Stop
Promoter
Fusion Proteins
6xHisGST
MycV5 rbs
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Do attB Sites Affect Expression in E. coli?
T7 lac O
RBS
topo
V5 6XHis
topopET 101 D-TOPO®
pET-DEST42 V5 6XHisatt B2
T7 lac O
att B1
pENTR SD/D-TOPO® att L1 RBS
topo
topo
att L2
RBS ORF
ORF
vs.
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Expression in Standard and Gateway®-Modified Vectors
GUS 6xHis-Gus GST-GUSTrx-GUS
U II I IU UU
Std GWStdGW Std GWStdGW
U II I IU UU
MW MW MW MW
50 kDa
E. coli strain BL21-SI U = Uninduced, I = Induced
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Expression levels of Human Kinases
ORF MW (kDa) pET-DEST42* pET 101 DT*H-U27143 14 + ++H-L25610 18 ++ ++H-M57730 23 +++ +H-X79483 37 - -H-D87116 38 ++ ++H-U02680 39 ++ +H-U00803 56 ++ ++H-M36881 56 ++ -H-M60724 58 ++ +++H-X52479 82 + +H-M80613 83 + +H-X97335 99 + +
*comparative expression levels are depicted in arbitrary units
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Expression of Full-Length Human ORFs
Baculovirus/Sf9 Insect Cells
4 5 6 7 81 2 3
E. coli strain BL21 SI
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50
220
1 2 3 4 5 6
Lane 1: 6xHis-GUSLane 2: 6xHis-MAP4Lane 3: 6xHis--AdaptinLane 4: 6xHis-Transferrin ReceptorLane 5: 6xHis-Tyr KinaseLane 6: 6xHis-EIF4e
kDa
Lane 1: GST-GUSLane 2: 6xHis-GUS Lane 3: GUSLane 4: MAP4Lane 5: -AdaptinLane 6: Transferrin ReceptorLane 7: Tyr Kinase Lane 8: EIF4e
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human p70 ribosomal S6 kinase
5 6 7 84321M M
u i u u ui i i
pET-DEST42 pET 101 DT
kDa
51
64
39
*58kd
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Female sterile homeotic protein
5 6 7 84321Mk Da
97
66
u u u ui i i i
pET-DEST42 pET 101 DT
*85 k Da protein
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Receptor tyrosine kinase ligand
5 6 7 84321Mk Da
u u u ui i i i
pET DEST 42 Gateway®
pET 101 D-TOPO®Non-Gateway®
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21
*23kd
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Gene Expression Mammalian Cells
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Two Entry Points for Expression
L1 L2
pENTR/D-TOPO®
B1 B2
pcDNA3.2/GW/D-TOPO®
ORF
V5
ORF
pENTR-ORF
L1 L2
R1 R2
pcDNA3.2-DEST
V5ccdB
B1 B2
pcDNA3.2 GW-ORF
V5ORF
LR
*You can also clone your PCR product directly into this vector bypassing entry clone construction
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pcDNA/GW/ D-TOPO® Vectors
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Expression of ORFs in CHO Cells
lacZ Gus
GS5 GS1
0 GS1
5 GS1
9 A3 B9
GFP GS2 GS7
120kD
80kD
20kD
30kD
50kD
1 2 3 4 5 6 7 8 9 10 11
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8 x 104 COS-7L cells, 0.8 g each DNA/ well, 24 h post-transfection
Lipofectamine™ 2000 Reagent (l)
1.0 1.5 2.0 2.5 3.0 3.5
pCMVneo-GUS
pCMV•SPORT-gal
Expression in Mammalian Cells
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