Recombinant DNA II

11/20/2008Biochem: Recombinant DNA

Recombinant DNA II

Andy HowardIntroductory Biochemistry

20 November 2008


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Recombinant DNA (review) Much of our current understanding of molecular biology, and of the ways we can use it in medicine, agriculture, and basic biology, is derived from the kinds of genetic manipulations that we describe as recombinant DNA


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What we’ll discuss Expression Genomics Proteomics Amplification

Polymerase Chain Reaction

Mutagenesis Random Site-directed

Applications Probing protein-protein interactions


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Using expression vectors We often want to do something with cloned inserts in expression vectors, viz. make RNA or even protein from it

RNA: stick an efficient promoter next to the cloning site; vector DNA transcribed in vitro using SP6 RNA polymerase

This can be used as a way of making radiolabeled RNA


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Protein expression Making (eukaryotic) proteins in bacteria via cDNA means we don’t have to worry about introns

Expression vector must have signals for transcription and translation

Sequence must start with AUG and include a ribosome binding site

Strong promoters can coax the bug into expressing 30% of E.coli’s protein output to be the one protein we want!


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Example: ptac

This is a fusion of lac promoter (lactose metabolism) with trp promoter (tryptophan biosynthesis)

Promoter doesn’t get turned on until an inducer (isopropyl--thiogalactoside, IPTG) is introduced

QuickTime™ and a decompressor

are needed to see this picture.


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Eukaryotic expression Sometimes we need the glycosylations and other PTMs that eukaryotic expression enables

This is considerably more complex Common approach is to use vectors derived from viruses and having the vector infect cells derived from the virus’s host

Example: baculovirus, infecting lepidopteran cells; gene cloned just beyond promoter for polyhedrin, which makes the viral capsid protein


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Screening libraries with antibodies

Often we have antibodies that react with a protein of interest

If we set up a DNA library and introduce it into host bacteria as in colony hybridization, we can put nylon membranes on the plates to get replicas of the colonies

Replicas are incubated to make protein Cells are treated to release the protein so it binds to the nylon membrane

If the antibody sticks to the nylon, we have a hit!


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Fusion proteins Sometimes it helps to co-express our protein of interest with something that helps expression, secretion, or behavior

We thereby make chimeric proteins, carrying both functionalities

We have to be careful to keep the genes in phase with one another!

Often the linker includes a sequence that is readily cleaved by a commercial protease


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Fusion systems (table 12.2+)

Product Origin

Mass,kDa

Secreted?

Affinity Ligand

-galactosidase

E.coli

116 No APTG

Protein A Staph.

31 Yes IgG

Chloramphenicolacetyltransferase

E.coli

24 Yes Chloram-phenicol

Streptavidin Strep.

13 Yes Biotin

Glut-S-tranferase

E.coli

26 No Glutathione

Maltose Bind.Prot.

E.coli

40 Yes Starch

Hemoglobin Vitreo-scilla

16/32

No None


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Improving purification via expression

If we attach (usually at the N-terminal end) a his-tag (several his, several cys) to our protein, it becomes easier to purify:

The his tag forms a loop that will bind strongly to a divalent cation like Ni2+

Thus we can pour our expressed protein through a Ni2+ affinity column and it will stick, while other proteins pass through

We elute it off by pouring through imidazole, which completes for the Ni2+

and lets our protein fall off


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Protein-protein interactions

One of the key changes in biochemistry over the last two decades is augmentation of the traditional reductionist approach with a more emergent approach, where interactions among components take precedence over the properties of individual components

Protein-protein interaction studies are the key example of this less determinedly reductionist approach


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Two-hybrid screens

Use one protein as bait; screen many candidate proteins to see which one produces a productive interaction with that one

Thousands of partnering relationships have been discovered this way

Some of the results are clearly biologically relevant; others less so


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2-hybrid screen

X is bait, fused to DNA binding domain of GAL4

Y is target, fused to transcriptional activator portion of GAL4


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Reporter constructs:How to study regulation Put a regulatory sequence into a plasmid upstream of a reporter gene whose product is easy to measure and visualize

Then as we vary conditions, we can see how much of the reporter gets transcribed

Example: Green Fluorescent Protein, which can be readily quantified based on fluorescent yield


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Genomics Application of these high-throughput techniques to identification of genetic makeup of entire organisms

First virus was completely sequenced in the late 1970’s

First bacterium: Haemophilus influenzae, 1995

Now > 50 organisms in every readily available phylum


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What’s been sequenced? Current list would be even longer

Also include multiple individuals within a species


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How genomics works A researcher who wishes to draw general conclusions about structure-function relationships may want to learn the sequence (“primary structure”) of many genes and non-genomic DNA in order to draw sweeping conclusions or build a library of genetic constructs, some of which he will understand and others he won’t


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Complete sequencing of a genome Fragment chromosomes Shotgun sequencing of fragments Reconstitution based on overlaps Cross-checking to compensate for errors

Interpretation


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Human genome project

Effort began in late 1980’s to do complete sequencing of the human genome

Methods development was proceeding rapidly during the period in question so it “finished” well ahead of schedule in 1999

Partly federal, partly private Related efforts in other countries


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What’s the point?

Better understanding of both coding and non-coding regions of chromosomes

Identification of specific human genes

Medically significant results Statistical results (x% are Zn fingers…)

Variability within Homo sapiens or some other sequenced organism by comparing complete sequences or ESTs between individuals


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Proteomics Analysis of the resulting list of expressible (not necessarily expressed!) proteins

Often focuses on changes in expression that arise from changes in environmental conditions or stresses

Often useful to analyze mRNAs along with proteins

Mass spectrometry is a key tool in proteomics


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How MS works in proteomics

Cartoon from Science Creative Quarterly at U.British Columbia, 2008




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Amplification Prokaryotic and eukaryotic cells can, through mitosis, serve as factories to make many copies (> 106 in some cases) of a moderately complex segment of DNA—provided that that segment can be incorporated into a chromosome or a plasmid

This is amplification


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Polymerase chain reaction

This is a biochemical tool that enables incorporation of desired genetic material into a cell’s reproductive cycle in order to amplify it

Start with denatured DNA containing a segment of of interest

Include two primers, one for each end of the targeted sequence

The sequence of events is now well-defined after three decades of refinement of the approach


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PCR: the procedure Heat to denature cellular dsDNA and separate the strands

Add the primers (ssDNA) and polymerase

Heat again, then cool enough for ligation

Continue cycling to get many cell divisions ~ 106-fold amplification


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PCR in practice


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RT-PCR Variant on ordinary PCR: starting point is an RNA probe that can serve as a template for DNA via reverse transcriptase

Once cDNA copy is available, normal PCR dynamics apply



Cartoon courtesy Cellular & Molecular Biology group at ncvs.org


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Mutagenesis Procedure through which mutations are introduced into genomic DNA

May be used: To generate diversity To probe the essentiality of specific genes

To examine particular segments of genes To alter properties of DNA or its mRNA transcript or a translated protein

To provide information and material for gene therapy


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Random mutagenesis DNA (often locally ssDNA) is exposed to mutagens in order to introduce random mispairings or increase the rate of mispairing during replication Can involve ionizing radiation Can involve chemical mutagens:

Error-prone PCR Using “mutator strains” Insertion mutagenesis Ethyl methanesulfonate Nitrous acid and other nitroso compounds


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Site-directed mutagenesis Specific loci in DNA targeted for alteration

Typically involves excision, addition of altered bases, and religation

Can be accomplished even in eukaryotic cell systems

Many biochemical systems can be systematically probed this way: To find essential amino acids in expressible proteins

To see which amino acids are important structurally

To examine changes at RNA level


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How do we use these tools?

Already discussed significance of complete sequencing efforts

Generally: amplification and expression give us access to and control of biochemical systems that otherwise have to be isolated in their original setting

These methods enable controlled experiments on complex systems


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Gene therapy Cloned variant of deficient gene is inserted into human cells

Can be done via viral or other vector carrying an expression cassette

Maloney murine leukemia virus works for cassettes up to 9kbp; depends on integrating the cassette into the patient’s DNA

Adenovirus works up to 7.5 kb: never gets incorporated into host, but simply replicates along with host


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Retroviral approach


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Adenoviral approach

Recombinant DNA II

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