RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Enzymes used in molecular cloning

2. Restriction endonucleases (REases) • Methylases • Ligases • Polymerases • Other nucleases • Kinases • Phosphatases • Topoisomerase

3. Biotech companies • New England Biolabs http://www.neb.com/nebecomm/default.asp • Fermentas http://www.fermentas.com • Promega http://www.promega.com • Stratagene http://www.stratagene.com • Merck Biosciences (Novagene) http://www.merckbiosciences.co.uk/home.asp • BD Biosciences (Clonetech, Pharmingen) http://www.bdbiosciences.com • Sigma-Aldrich http://www.sigmaaldrich.com • GE Healthcare (Amersham Biosciences, Pharmacia) http://www.gehealthcare.com/usen/index.html

4. Fermentas is a Lithuanian company

5. RESTRICTION AND MODIFICATION EXPERIMENTS (DUSSOIX & ARBER, 1962) • E coli K and C strains • Bacteriophage lambda (l) 1010 pfu/1ml Phage does not absorb to „new” host – false Phage DNA does not enter into „new” host – false Phage DNA rapidly degraded in „new” host – TRUE RESTRICTION ENDONUCLEASES METHYLASES

7. Restriction endonucleases http://www.typei-rm.info/index.htm http://www.typei-rm.info/index.htm

8. EcoR124I recognises GAAn6RTCG where R = purine and n = any nucleotide

9. Assembly into an anti-codon nuclease (ACNase) The bacteriophage T4 encodes a small (26 amino acid) polypeptide called Stp, which can interact with the EcoprrI restriction endonuclease as an anti restriction determinant The EcoprrI restriction endonuclease interacts with another host protein, PrrC, to produce a latent anti-codon nuclease (ACNase). The fully functional ACNase is able to cleave tRNALys at the codon triplet. however, to prevent cleavage of host (bacterial) tRNA the enzyme is latent and shows no such activity. Infection of the bacteria by bacteriophage T4 introduces the Stp polypeptide as an early gene and this polypeptide can activate the ACNase resulting in cleavage of the bacteriophage tRNALys. This prevents growth of the bacteriophage

10. Restriction endonucleases

11. Restriction endonucleases

12. Type II restriction endonucleases • >3500 type II enzyme,(750 strains) • Rebase enzyme list: >10,000 • 292 sequenced recognition seqence • REBASE(New England Biolabs): • http://rebase.neb.com/rebase/rebase.charts.html • Nomenclature: • HindIII Haemophilus influenzae dserotype, 3. enzyme • SmaI Serratia marcescens 1. enzyme

13. Type II restriction endonucleasesMore detailed classification

14. Type II restriction endonucleases • Recognition sequence: • Usually palindrome • Terta-, penta-, hexa-, octanucleotide • HpaIICCGG • HinfIGANTC • BamHIGGATCC • NotIGCGGCCGC • Interrupted palindrom • BstXI CCANNNNNTGG

15. Type II restriction endonucleases • Cleavage within the recognition sequence • In the middle: blunt • EcoRV GATATC • Asymetrically: sticky • 5’ overhang EcoRIGAATTC • 3’ overhang KpnIGGTACC • length of overhang: 1, 2, 3, 4, (5) • Frequency of recognition sites • Ptetra=4-4 =1/256 • Phexa=4-6 =1/4096

16. Type II restriction endonucleases

17. Type II restriction endonucleases W=A,T M=A,C R=A,G S=C,G Y=C,T K=G,T V=A,C,G H=A,C,T D=A,G,T B=C,G,T

18. EcoRI Type II restriction endonucleases

19. KpnI Type II restriction endonucleases

20. Type II restriction endonucleases recongize and cut only double stranded DNA • HindIII co-crystalized with CCAAGCTTGG • the strands are not separated • „indirect readout” • pattern in the major groove read by the enzyme

21. Type II restriction endonucleases • Isoschizomers • identical recognition and cleavage sites MboI, Sau3AIGATC • Neoschizomers • identical recognition, different cleavage SmaI CCCGGG XmaI CCCGGG • Compatible overhangs MboIGATC BamHGGATCC BglIIAGATCT BclITGATCA

22. Type II restriction endonucleases • Effect of methylation • Methylases in E. coli: • DammethylaseGmN6ATC GmN6ATC MboI – sensitive Sau3AI, Bsp143I – cleaves TCTAG mN6A XbaI – sensitive if overlapping dam site • DcmmethylaseCm5CWGG Cm5CGG HpaII – sensitive MspI – cleaves DpnI – cleaves only methylated GGGCCm5C ApaI – sensitive if overlapping dcm site • CpGmethylation in eukaryots SmaI - sensitive

23. Conditions for digestion Optimal buffer (pH, I, composition) Temperature (not always 37oC!) Star activity (relaxation of specificity) High glycerol conc. Excess enzyme Low ionic strength, high pH etc. Site preferences Cleavage rates differing by an order of magnitude Activity: 1unit cleaves 1 g in 60 min under optimal conditions (in 50 l) Cleavage close to the end (see tables in catalogs!) Double digestion (see optimal buffers in catalogs!) Type II restriction endonucleases

24. Cleavage close to the end http://www.fermentas.com/techinfo/re/restrdigpcrii.htm

25. Double digestion http://www.fermentas.com/doubledigest/index.html

26. Methylases EcoRI methylase

27. Ligases

28. Ligases

29. Ligases

30. T4 RNA ligase

31. DNA polymerases DNA pol I Klenow enzyme T4 polymerase T7 polymerase Thermostable Taq polymerase Pfu polymerase Reverse transcriptase

32. 3’ 5’ nuclease activity basicly proofreading activity – improves fidelity in the absence dNTP-s slowly removes correct base pairs too T4 polymerase active on 3’ overhang: generates blunt end: „polishing”

33. 5’->3’ nuclease activity Nick translation!

34. Reverse transcriptase

35. RNase activity associated with RT

37. T7 T3 Sp6 RNA polymerase run off synthesis

38. Template independent polymerases • DNA • Terminal deoxyribonucleotidyl transferase • Template: single stranded DNA • 3’ overhang • Co2+ : blunt, 5’ overhang, RNA • Homopolymer tailing • RNA • Poly(A) polymerase • Labeling 3’ ends of RNA

39. Terminal transferase

40. Polynucleotide kinase

41. Polynucleotide kinase: exchange reaction

42. Phosphatases Cut phosphate ester bonds • Bacterial alkaline phosphatase (BAP) • Calf intestinal phosphatase (CIP) • Shrimp phosphatase