Purificazione di proteine umane da animali

1. Purificazione di proteine umane da animali • Basse rese • Difficili da purificare • Costoso • Possibilita’ di malattie

2. How can we synthesise human proteins? • Use bacterial cells • Human gene lacks • Bacterial promoter • Bacterial terminator • Bacterial ribosome binding site • Cannot deal with introns

3. DNA RNA Reverse transcriptase RNA Protein DNA Dealing with introns

4. Inexpensive Easy to manipulate Well characterized Grows quickly rProtein up to 50% total protein Post-transcriptional modification Post-translational modification Poor folding Proteolysis N-terminal Methionine Complicated purification Lack of efficient secretion Possible toxicity Protein Expression in E. coli Advantages and Disadvantages

5. Promoter Selectable Marker E. coli Expression Vector

6. E. coli Promoters

7. Weickert, et al., 1996

8. SD AUG Promoter Stop Transcriptional Terminator Repressor Selectable Marker Selectable Marker Ori E. coli Expression Vector E. coli Expression Vector Promoter

9. What if expression is low? Optimizing Expression • Examine codon usage • Decrease message stability • Premature termination of transcription • Premature termination of translation • Frameshifts, deletions, and misincorporation

10. Codon Frequency in E. coli

11. What if expression is low? Optimizing Expression • Combined approach • Examine codon usage • Minimize GC at 5’ • Add terminator • Add fusion and/or tags • Growth conditions

12. Expression of Fusion Proteins • Ease of detection • Increase solubility • Increase stability • Increase expression • Ease of purification

13. Hexahistidine-tag GST MBP CBP/Intein Arg-tag S-tag Ni affinity GSH Amylose Chitin Ion-Exchange RNAse Examples of Fusions/Tags

14. Insoluble Proteins • Growth Temp • Media • Expression rate • Chaperones • Coexpression of subunits • Express as polymer • Redox potential • Periplasmic expression • Fusion • Tags • Express as a fragment • Denature and renature • Combined approach

15. Improving Protein Stability • Protease inhibitors • Protease-minus host • Periplasmic expression • Growth temperature • Combined approach

16. MANIPOLAZIONE DELL’ESPRESSIONE GENICA NEI PROCARIOTI -PROTEINE DI INTERESSE TERAPEUTICO E COMMERCIALE POSSONO ESSERE PRODOTTE IN E. coli CON TECNICHE DNA RICOMBINANTE -PROMOTORE -SEQUENZE LEGANTI I RIBOSOMI ( 6-8 nt Seq. di Shine Dalgarno) -NUMERO COPIE DEL GENE CLONATO -LOCALIZZAZIONE FINALE PROTEINA -STABILITA’ PROTEINA IN CELLULA OSPITE

17. GENI IN PROCARIOTI POSSONO AVERE -ESPRESSIONE COSTITUTIVA -ESPRESSIONE REGOLATA (es. lac operon) NELLA PRODUZIONE DI PROTEINE ETEROLOGHE IN BATTERI VENGONO UTILIZZATI SPESSO PROMOTORI FORTI E REGOLABILI UNA PRODUZIONE CONTINUA PROVOCA: -INIBIZIONE FUNZIONI CELLULA -PERDITA ENERGIA -PERDITA PLASMIDE

18. Bottlenecks to efficient protein expression in E. coli l Inefficient transcription No or little protein synthesized u Promoter choice and design l Inefficient translation No or little protein synthesized u Codon usage Transcript stability Transcript secondary structure u u Aggregation or degradation l Inefficient folding (cytoplasmic or periplasmic) u Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation Inefficient isomerization of peptidyl-prolyl bonds u u Aggregation or degradation l Inefficient membrane insertion/translocation l Toxicity Cell death

19. J K K T F J ATP 3' 5' GrpE ATP GrpE ADP ADP Native Aggregate ADP ATP GroEL GroES Folding chaperones in de novo folding

20. GroEL-GroES co-expression and low temperatures improve leptin folding

21. However, this strategy does not always work

22. cDNA di interesse MCS MBP o GST PROTEINE DI FUSIONE -PER EVITARE DEGRADAZIONE DI PICCOLE PROTEINE ETEROLOGHE QUESTE VENGONO PRODOTTE COME PROTEINE DI FUSIONE CON UNA PROTEINA STABILE DELL’ORGANISMO OSPITE. -I DUE cDNA DEVONO ESSERE FUSI MANTENENDO LA CORRETTA CORNICE DI LETTURA PROMOTORE REGOLABILE

23. cDNA di interesse MCS MBP o GST GST o MBP UTILIZZATE PER PURIFICAZIONE SITO DI TAGLIO PER PROTEASI PROTEINA DI INTERESSE INDUZIONE DI ESPRESSIONE PROTEINA DI FUSIONE (PROMOTORI REGOLABILI) TRASFORMAZIONE IN BATTERI

24. Promotore “lac” gene MalE Proteina di fusione MBP pMAL cDNA di interesse Resina con legato maltosio Eluizione Proteina di fusione purificata -

25. pGEX GST comes from Schistosoma mansoni Foreign gene GST • IPTG • induction • High level • expression tac

26. PURIFICATION OF GST FUSION PROTEINS

27. PURIFICATION • EASY • AFFINITY CHROMATOGRAPHY

28. PURIFICATIONDETAILS • GROW SAY 1L CULTURE TO MID LOG PHASE • ie OD260 = 0.4 – 0.7 • SPIN DOWN CELLS • SONICATE IN PRESENCE OF PROTEASE INHIBITORS • POUR LYSATE OVER GLUTAHIONE SEPHAROSE BEADS IN A COLUMN

29. glutathione SEPHAROSE GLUTATHIONE SEPHAROSE

30. FUSION PROTEIN FOREIGN PEPTIDE GST

31. FOREIGN PEPTIDE GST glutathione SEPHAROSE FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE

32. PURIFICATION • WASH COLUMN EXTENSIVELY • ELUTE WITH REDUCED GLUTATHIONE • RESULTS IN PURE GST FUSION PROTEIN

33. COMPETITIVE ELUTION WITH GLUTATHIONE SEPHAROSE

34. pure fusion protein + glutathione foreign peptide + GST protease dialyse second glutathione column pure fusion pure foreign peptide in flow through - GST sticks RESULT OF AFFINTY PURIFICATION AND REMOVAL OF GST MOIETY

35. pQE VECTORS (Qia Express) • Hex-histidine tag system • Produce peptides with 6 histidines fused to N or C terminus • Allows Nickel Chelate Affinity Chromatography

36. pQE VECTORS (Qia Express) • Promoter • engineered from phage T5 + lac operator • 2 operator sites • IPTG inducible • Expression in host containing multiple copies of pREP4 which has lacI

37. pQE VECTORS (Qia Express) • Interaction between Ni2+ resin called NTA is very strong and chemically resilient • every Ni2+ binds 2 his residues in a non-conformation dependent manner • therefore resists strong denaturants eg 6M guanidium HCl

38. pQE VECTORS (Qia Express) • Elution • competitive with imidazole

39. pQE VECTORS (Qia Express) • Removal of His tag? • not necessary usually • many hundreds of proteins purified with no effect on structure • not immunogenic

40. PROTEINE DI INTERESSE TERAPEUTICO IN PROCARIOTI: -RISCHIO CONTAMINAZIONE VIRALE NULLO -RISCHIO ALLERGIE NULLO (vengono prodotte proteine umane) PRODUZIONE DI INSULINA UMANA IN E. coli -70 MAIALI PER 1 PAZIENTE PER UN ANNO -E. Coli NON SA MODIFICARE premRNA EUCARIOTICI E PRODURRE MODIFICHE POST-TRASCRIZIONALI

41. SINTESI INSULINA IN CELLULA PANCREATICA CATENA A 30 aa CATENA B 21 aa Unite da ponti S-S ESONE 2 ESONE 1 PREPROINSULINA PEPTIDE SEGNALE PROINSULINA FORMA S-S IN APPARATO DEL GOLGI UN ENZIMA RIMUOVE 33aa INSULINA

42. PRODUZIONE DI INSULINA RICOMBINANTE IN BATTERI -Plasimidi separati codificano per Catena A e B -promotore trp e alcuni codoni iniziali trp -seq per il trp sono eliminate con trattamento con bromuro di cianato -catene mescolate assieme e tramite un processo chimico si formano legami S-S

43. PRODUZIONE ORMONE DELLA CRESCITA UMANO IN E. Coli -Peptide di 191 aa -Carenza provoca nanismo -GH da animali non è efficace sull’uomo -80 ipofisi di cadaveri umani per un paziente per un anno (alto rischio infezioni)

44. PRODUZIONE DI GH RICOMIBINATE IN BATTERI

45. SALMONELLA • Expression host • Live vaccine delivery

46. SALMONELLA • Salmonella is itself a pathogen – S.typhi causes typhoid • It is possible to vaccinate aganst with attenuated strains • Attenuated Salmonella can persist in the gut and disseminate • Induces mucosal & systemic cellular & humoral responses • It has potential to be engineered as one shot, multivalent vaccines

47. SALMONELLA • Recognises E.coli promoters and origins of replication • therefore existing vectors can function • Several ways of attenuating Salmonella have been discovered

48. EXPRESSION SYSTEMS MOST USE PLASMIDS • PROBLEMS • INSTABILITY • TOXICITY • pIP-pET DUAL PLASMID • NirB-ANAEROBIC INDUCIBLE • BALANCED LETHAL

49. foreign antigen T7 promoter pET AmpR pIP pIP-pET DUAL PLASMID pL c1ts T7 RNA polymerase kanR c1ts= l repressor active 28°C, inactive at 37°C pL = l left promoter

50. pTECH VECTORS • THESE USE THE NIRB PROMOTER • NIRB ENCODES NADH-DEPENDENT NITRITE REDUCTASE • NIRB INDUCED IN ANAEROBIC CONDITIONS eg GUT & TISSUES