Study and engineering of gene function: mutagenesis

1. Study and engineering of gene function: mutagenesis • Why mutagenize? • Random mutagenesis, mutant selection schemes • Site-directed mutagenesis, deletion mutagenesis • Engineering of proteins • Alterations in the genetic code Course Packet: #30

2. Uses for mutagenesis • Define the role of a gene--are phenotypes altered by mutations? • Determine functionally important regions of a gene (in vivo or in vitro) • Improve or change the function of a gene product • Investigate functions of non-genes, eg. DNA regions important for regulation

3. Protein engineering-Why? • Enhance stability/function under new conditions • temperature, pH, organic/aqueous solvent, [salt] • Alter enzyme substrate specificity • Enhance enzymatic rate • Alter epitope binding properties

4. Enzymes: Biotech Cash Crops

5. Obtaining useful enzymes From Koeller and Wang, “enzymes for chemical synthesis”, Nature 409, 232 - 240 (2001)

6. Random mutagenesis • Cassette mutagenesis with “doped”oligos • Chemical mutagenesis • expose short piece of DNA to mutagen, make “library” of clones, test for phenotypes • PCR mutagenesis by base misincorporation • Include Mn2+ in reaction • Reduce concentration of one dNTP

7. Random mutagenesis by PCR: the Green Fluorescent Protein Screen mutants

8. Cassette mutagenesis (semi-random) Translation of sequence Strands synthesized individually, then annealed Allows random insertion of any amino acid at defined positions

9. Random and semi-random mutagenesis: directed evolution • Mutagenize existing protein, eg. error-prone PCR, doped oligo cassette mutagenesis -- and/or -- Do “gene shuffling” (Creates Library) • Screen library of mutations for proteins with altered properties • Standard screening: 10,000 - 100,000 mutants • Phage display: 109 mutants

10. Gene shuffling: “sexual PCR”

11. Gene shuffling For gene shuffling protocols you must have related genes in original pool: 1) evolutionary variants, or 2) variants mutated in vitro Shuffling allows rapid scanning through sequence space: faster than doing multiple rounds of random mutagenesis and screening

12. Shuffling of one gene mutagenized in two ways

13. Gene shuffling--cephalosporinase from 4 bacteria Single gene mutagenesis Multiple gene shuffling

14. Screening by phage display: create library of mutant proteins fused to M13 gene III Random mutagenesis Human growth hormone: want to generate variants that bind to hGH receptor more tightly

15. Phage display:production of recombinant phage The “display”

16. Phage display: collect tight-binding phage The selection

17. Animation of phage display http://www.dyax.com/discovery/phagedisplay.html

18. Site-directed mutagenesis: primer extension method Drawbacks: -- both mutant and wild type versions of the gene are made following transfection--lots of screening required, or tricks required to prevent replication of wild type strand -- requires single-stranded, circular template DNA

19. Alternative primer extension mutagenesis techniques

20. “QuikChangeTM” protocol Destroys the template DNA (DNA has to come from dam+ host Advantage: can use plasmid (double-stranded) DNA

21. First PCR Site-directed mutagenesis: Mega-primer method A Second PCR Wild type template B Megaprimer needs to be purified prior to PCR 2 Allows placement of mutation anywhere in a piece of DNA

22. Domain swapping using “megaprimers” (overlapping PCR) -C N- Template 1 PCR 1 Mega-primer Template 2 PCR 2 Domains have been swapped

23. PCR-mediated deletion mutagenesis Target DNA PCR products Oligonucleotide design allows precision in deletion positions

24. Directed mutagenesis • Make changes in amino acid sequence based on rational decisions • Structure known? Mutate amino acids in any part of protein thought to influence activity/stability/solubility etc. • Protein with multiple family members? Mutate desired protein in positions that bring it closer to another family member with desired properties

25. An example of directed mutagenesis T4 lysozyme: structure known Can it be made more stable by the addition of pairs of cysteine residues (allowing disulfide bridges to form?) without altering activity of the protein?

26. T4 lysozyme: a model for stability studies Cysteines were added to areas of the protein in close proximity--disulfide bridges could form

27. More disulfides, greater stabilization at high T Bottom of bar: melting temperature under reducing condtions Top of bar: Melting temperature under oxidizing conditions Green bars: if the effects of individual S-S bonds were added together

28. Stability can be increased - but there can be a cost in activity

29. The genetic code • 61 sense codons, 3 non-sense (stop) codons • 20 amino acids • Other amino acids, some in the cell (as precursors to other amino acids), but very rarely have any been added to the genetic code in a living system • Is it possible to add new amino acids to the code? • Yes...sort of Wang et al. (2001) “Expanding the genetic code” Science292, p. 498.

30. Altering the genetic code

31. Why add new amino acids to proteins? • New amino acid = new functional group • Alter or enhance protein function (rational design) • Chemically modify protein following synthesis (chemical derivitization) • Probe protein structure, function • Modify protein in vivo, add labels and monitor protein localization, movement, dynamics in living cells

32. How to modify genetic code? • Adding new amino acids to the code--must bypass the fidelity mechanisms that have evolved to prevent this from occurring 2 key mechanisms of fidelity • Correct amino acid inserted by ribosome through interactions between tRNA anti-codon and mRNA codon of the mRNA in the ribosome • Specific tRNA charged with correct amino acid because of high specificity of tRNA synthetase interaction • Add new tRNA, add new tRNA synthetase

33. tRNA charging and usage Charging: (tRNA + amino acid + amino acyl-tRNA synthetase) Translation: (tRNA-aa + codon/anticodon interaction + ribosome)

34. Chose tRNAtyr, and the tRNAtyr synthetase (mTyrRS) from an archaean (M.jannaschii)--no cross-reactivity with E. coli tRNAtyr and synthetase • Mutate m-tRNAtyr to recognize stop codon (UAG) on mRNA • Mutate m-TyrRS at 5 positions near the tyrosine binding site by doped oligonucleotide random mutagenesis • Obtain mutants that can insert O-methyl-L-tyrosine at any UAG codon

36. Outcome • Strategy allows site specific insertion of new amino acid--just design protein to have UAG stop codon where you’d like the new amino acid to go • Transform engineered E. coli with plasmid containing the engineered gene • Feed cells O-methyl tyrosine to get synthesis of full length gene

37. Utility of strategy • Several new amino acids have been added to the E. coli code in this way, including phenyalanine derivatives with keto groups, which can be modified by hydrazide-containing fluorescent dyes in vivo • Useful for tracking protein localization, movement, and dynamics in the cell p-acetyl-L-phenylalanine m-acetyl-L-phenylalanine

38. Some questions: • What are the consequences for the cell with an expanded code? • Do new amino acids confer any kind of evolutionary advantage to organisms that have them? (assuming they get a ready supply of the new amino acid…) • Why do cells have/need 3 stop codons????