RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Cloningstrategies;mutagenesis

2. RECOMBINANT CONSTRUCTIONS • INSERT • A) genomic DNA fragments(generatedbyrestrictionendonucleaseormechanicfragmentation) • B.) cDNS („complementary” DNA copy of an mRNA), • C.) PCR product • DNA isolationusuallyfromagarosegel; • LIGATION„theoreticalligation” (bimolecularreactionvs. circularisation) effective end concentration:j=51,1xMr½ (e.g. jpBR322=32 mg/ml) c<j circularisation; c>j concatamerisation ifci/cv≈2-3yieldof recombinantmolecule is max 40%

3. Ligation is a tricky reaction Lane (i) contains a molecularweight ladder (1 Kb ladder, Bethesda Research Labs). Lanes (a) through (h) were ligatedat, respectively, 1.6, 3.1, 6.2, 12.5, 25, 50, 100, and 200 mg/mI

4. Cloning strategies • Cohesiveends- compatible enzymes • Single enzyme(self closing of the vector – CIP treatment) • Two enzymes - directional cloning • Linkers:pCGGATCCG BamHI • Adapters:AATTCGCGGCCGCEcoRI -NotI • GCGCCGGCGp • Blunt ends can be ligated if there are no compatible restriction sites • Filling of 5’ overhang (any polymerase) • Cut back of 3’ overhangs – exonuclease –T4 polymerase

5. CIP TREATMENT

6. Cloning strategies Topo cloning

8. Cloning strategies LIC – ligation independent cloning ~12 bp complementary overhang, limited ExoIII digestion overhang generation by T4 polymerase

9. Cloning strategies LIC – ligation independent cloning Here we report a rapid and simple method for LIC-PCR using primers containing the nonbase residue 1,3-propanediol in defined positions. Clonable complementary ends are produced directly in the PCR when Taq DNA polymerase stops at the nonreplicable element leaving the rest of the primer single stranded: X = 1,3-propanediol, modified NotI recognition site is in bold.

10. Cloning by recombination

14. Introducing DNA in prokaryiotic cells • transformation (=transfection): plasmid,competentE. coli chemical treatment CaCl2, MgCl2, MnCl2, hexamin-CoCl3, DMSO glycerol -80 C 105-108colony/g superhelical DNA) • infection: • bakteriophage + permissivehost • elektroporation: electric shock (~15 kV/cm) (1010colony/g superhelical DNA) salt free DNA!!!!

15. Study and engineering of gene function: mutagenesis • Why mutagenize? • Random mutagenesis, mutant selection schemes • Site-directed mutagenesis, deletion mutagenesis • Engineering of proteins

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

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

18. Enzymes: Biotech Cash Crops

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

20. 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

21. Random mutagenesis by PCR: the Green Fluorescent Protein Usually combined with DNA shuffling Screen mutants

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

23. 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

24. Gene shuffling: “sexual PCR”

25. 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

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

28. Shuffling of one gene mutagenized in two ways

29. 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

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

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

32. Site-directed mutagenesis: primer extension method Drawbacks: -- both mutant and wild type versions of the gene are present following transfection -- methylation dependent mismatch repair reduces the number of mutants --lots of screening requiredto prevent replication of wild type strand -- requires single-stranded, circular template DNA (M13 phage DNA) Solutions: Mutant E.coli strains -- deficient of mismatch repair genes

33. Kunkel method • Why there is no uracil in DNA? • Uracil spontaneously formed from citosine: G A transition mutation! • Every cell has a complex repair system: DNA uracil N-glycosidase: ung deoxyuridyl transferase: dut • DNA produced in ung-, dut- cells contains uracil instead of some thymines • ung+ cells degrade wild type methylated strand

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

35. Alternative primer extension mutagenesis techniques

37. Position of mutation gene gene gene gene vector vector vector vector vector vector vector vector Mutagenesis with PCR • Modification at the ends of amplicons • Adding restriction sites • Fusion of gene segments (see overlap extension) • Megaprimer method: 1st PCR 2nd PCR

38. Position of mutation gene gene gene gene vector vector vector vector vector vector vector vector Mutagenesis with PCR • Overlap extension 1st PCR 2nd PCR 3rd PCR

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

40. 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

41. 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?

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

43. 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

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

45. 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.

46. Altering the genetic code

47. 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

48. 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

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

50. Chose tRNAtyr, and the tRNAtyrsynthetase (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