Recombinant DNA II

1. Recombinant DNA II Andy HowardIntroductory Biochemistry 20 November 2008 Biochem: Recombinant DNA

2. 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 Biochem: Recombinant DNA

3. Expression Genomics Proteomics Amplification Polymerase Chain Reaction Mutagenesis Random Site-directed Applications Probing protein-protein interactions What we’ll discuss Biochem: Recombinant DNA

4. 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 Biochem: Recombinant DNA

5. 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! Biochem: Recombinant DNA

6. 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 Biochem: Recombinant DNA

7. 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 Biochem: Recombinant DNA

8. 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! Biochem: Recombinant DNA

9. 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 Biochem: Recombinant DNA

10. Fusion systems (table 12.2+) Biochem: Recombinant DNA

11. 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 Biochem: Recombinant DNA

12. 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 Biochem: Recombinant DNA

13. 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 Biochem: Recombinant DNA

14. 2-hybrid screen • X is bait, fused to DNA binding domain of GAL4 • Y is target, fused to transcriptional activator portion of GAL4 Biochem: Recombinant DNA

15. 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 Biochem: Recombinant DNA

16. 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 Biochem: Recombinant DNA

17. What’s been sequenced? • Current list would be even longer • Also include multiple individuals within a species Biochem: Recombinant DNA

18. 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 Biochem: Recombinant DNA

19. Complete sequencing of a genome • Fragment chromosomes • Shotgun sequencing of fragments • Reconstitution based on overlaps • Cross-checking to compensate for errors • Interpretation Biochem: Recombinant DNA

20. 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 Biochem: Recombinant DNA

21. 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 Biochem: Recombinant DNA

22. 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 Biochem: Recombinant DNA

23. How MS works in proteomics • Cartoon from Science Creative Quarterly at U.British Columbia, 2008 Biochem: Recombinant DNA

24. 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 Biochem: Recombinant DNA

25. 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 Biochem: Recombinant DNA

26. 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 Biochem: Recombinant DNA

27. PCR in practice Biochem: Recombinant DNA

28. 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 Biochem: Recombinant DNA

29. 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 Biochem: Recombinant DNA

30. 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 Biochem: Recombinant DNA

31. 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 Biochem: Recombinant DNA

32. 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 Biochem: Recombinant DNA

33. 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 Biochem: Recombinant DNA

34. Retroviral approach Biochem: Recombinant DNA

35. Adenoviral approach Biochem: Recombinant DNA