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. Dealing with introns DNA RNA Protein RNA DNA Reverse transcriptase

4. Protein Expression in E. coli 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 Advantages and Disadvantages

5. E. coli Expression Vector Selectable Marker Promoter

6. E. coli Promoters

7. Weickert, et al., 1996

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

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

10. Codon Frequency in E. coli

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

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

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

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 Promoter choice and design Inefficient transcriptionNo or little protein synthesized Codon usage Transcript stability Transcript secondary structure Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation Inefficient isomerization of peptidyl-prolyl bonds Inefficient translationNo or little protein synthesized Inefficient folding (cytoplasmic or periplasmic) Inefficient membrane insertion/translocation ToxicityCell death l l l l l u u u u u u u Aggregation or degradation

19. Folding chaperones in de novo folding Aggregate 3' 5' K TF J Native K ADP GrpE J GroEL GroES ATP ADP ATP ADP GrpE

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

21. However, this strategy does not always work

22. 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 MCS cDNA di interesse MBP o GST PROMOTORE REGOLABILE

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

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

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

26. PURIFICATION OF GST FUSION PROTEINS

27. PURIFICATION EASY AFFINITY CHROMATOGRAPHY

28. PURIFICATION DETAILS 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 GST FOREIGN PEPTIDE

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

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

33. COMPETITIVE ELUTION WITH GLUTATHIONE SEPHAROSE

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

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

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

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

51. pTECH VECTORS NirB promoter pTECH GST AmpR tetanus toxoid Khan made this vector Oral immunisation, single dose in mice -protected against Salmonella Tetanus toxin

52. BALANCED – LETHAL SYSTEM OTHER SYSTEMS DESCRIBED CARRY ANTIBIOTIC RESISTANCE-UNDESIREABLE THESE VECTORS COMPLEMENT LETHAL DELETION IN HOST GENE FOR B-ASPARTATE SEMI-ALDEHYDE DEHYDROGENASE OR asd asd MUTANTS HAVE ABSOLUTE REQUIREMENT FOR DIAMINOPIMELIC ACID (DAP) A CONSTITUENT OF THE CELL WALL THERE IS NO DAP IN MAMMALS

53. Balanced Lethal trc promoter pYA292 foreign gene asd asd complements asd  host & is thus stable

54. Heterologous Expression in Yeast Codon usage is closer to human Glycosylation of exported proteins Purification of proteins from the medium Ease of transformation Ease of growth

55. EXPRESSION IN PICHIA PASTORIS

56. PICHIA PASTORIS USES ALCOHOL OXIDASE 1 (AOX1) PROMOTER AOX1 IS INDUCIBLE BY METHANOL AND GENE IS EXPRESSED AT VERY HIGH LEVELS THERE ARE THREE BASIC STEPS

57. STEP1 CLONE GENE OF INTEREST INTO SHUTTLE VECTOR DOWNSTREAM OF AOX1 PROMOTER IN E. coli AOX1 promoter gene of interest TT HIS4+ 3’ AOX1

58. STEP2 TRANSFORM HIS - PICHIA PASTORIS YEAST WITH PLASMID. SELECT FOR HIS+ STABLE INTEGRANTS DISRUPTED IN THE AOX1 LOCUS

59. STEP2 AOX1 promoter gene of interest TT HIS4+ 3’ AOX1 gene of interest pAOX1 TT INTEGRATION P.pastoris chromosome

60. Pichia pastoris production of single-chain antibody fragments (scFv) A CASE STUDY 1. PLACE scFv cDNA in vector pPIC9K

61. pPIC9K pAOX1 scFv cDNAHis 6 tag  -mating type secretion signal PLACE scFv cDNA in vector pPIC9K ALL RECOMBINANT STEPS DONE IN E.coli

62. scFv expression in P. pastoris 2. Transform HIS - P. pastoris by electroporation Select on minimal media 3.Check medium for product after methanol induction. POSITIVE

63. scFv expression in P. pastoris 4. Large scale up 5 litres capacity stirred reactor 4L medium plus 400 ml starter culture Grow 17h @ 30 o C in glycerol Dense Keep pH stable @ 6.0 Induce 48 h with methanol Harvest culture medium Adjust pH to 7.4 and Affinity Purify by Nickel Chelate Chromatography

64. YIELDS For scFV antibody 250 mg per L OTHER EXAMPLES highest yield –tetanus toxin frag C 12g per L (INTRACELLULAR) –  amylase 2.5g per L (SECRETED) CAN WORK ON INDUSTRIAL SCALE

65. YIELDS PRODUCTYIELD g per L ENZYMES Invertase2.3  amylase 2.5 ANTIGENS Pertussis Antigen P603.0 Tetanus toxin fragment C12.0 HIV gp1201.25 Tick antigen1.5 CYTOKINES TNF10.0 Interferon alpha0.4 PROTEASES Carboxypeptidase B0.8 ANTIBODIES Rabbit single chain Fv0.25

66. ADVANTAGES OF EXPRESSION IN P. pastoris EUKARYOTE- some post-translational modification MICRO-ORGANISM –easy to manipulate –cheap YEAST – advanced molecular genetics HIGH YIELDS

67. Molecular Farming 1.A new field where plants and animals are genetically engineered to produce important pharmaceuticals, vaccines, and other valuable compounds. 2.Plants may possibly be used as bioreactors to mass-produce chemicals that can accumulate within the cells until they are harvested. 3.Soybeans have been used to produce monoclonal antibodies with therapeutic value for the treatment of colon cancer.

68. Molecular Farming 4.Plants have been engineered to produce human antibodies against HIV 5.Pharmaceuticals has begun clinical trials with herpes antibodies produced in plants. 6.The reasons that using plants may be more cost-effective than bacteria: a)Scale-up involves just planting seeds. b)Proteins are produced in high quantity. c)Foreign proteins will be biologically active. d)Foreign proteins stored in seeds are very stable. e)Contaminating pathogens are not likely to be present.

69. Molecular Farming Edible Vaccines a)People in developing countries have limited access to many vaccines. b)Making plants that produce vaccines may be useful for places where refrigeration is limited. c)Potatoes have been studied using a portion of the E. coli enterotoxin in mice and humans. d)Other candidates for edible vaccines include banana and tomato, and alfalfa, corn, and wheat are possible candidates for use in livestock. e)Edible vaccines may lead to the eradication of diseases such as hepatitis B and polio.

73. For the last decade, scientists have known how to genetically engineer a plant to produce a desired protein. The two most common tools used to do this are: Agrobacteria have a circular form of DNA called plasmids. The plasmids are easily manipulated because they naturally have two “cut” points where a gene can be taken out and replaced with one of the scientist’s choice. DNA is coated on microscopically tiny gold beads that are placed in a vacuum chamber. The gene gun then allows compressed gas to expand, pushing the beads down until they hit a filter. The DNA then flies off of the beads down into the tissue, where some will enter a nucleus and become incorporated. Cut out the selected region of the plasmid. Add the desired gene. Grow the plant like a regular crop. Infect the plant with the agrobacteria and grow it in a medium.

74. The plants that produce the edible vaccines could be grown in third world countries. Growing plants is much cheaper than producing vaccines. Plants are already regularly used in pharmaceuticals, so there are established purification protocols. Agricultural products can be transported around the world relatively cheaply. Plants can’t host most human pathogens, so the vaccines won’t pose dangers to humans.

75. Plants are living organisms that change, so the continuity of the vaccine production might not be guaranteed. Glycosylation patterns in plants differ from those in humans and could affect the functionality of the vaccines. If the vaccines were grown in fields or on trees, security would become a big issue. The dosage of the vaccines would be variable. For example, different sized bananas would contain different amounts of vaccine. The edible vaccines could be mistaken for regular fruits and consumed in larger amounts than might be safe.

77. Why HEK.EBNA Cells? The Principle integrated Ad5 E1a/E1b fragment in HEK 293 cells enhances trans- cription of CMV promotor driven transgene EBNA-1 protein drives episomal replication of ori-P containing plasmids EBNA-1/ori-P based expression in Human Embryonic Kidney (293) cells (293 stably transformed with EBNA-1 gene) The cell line is available from ATCC and, until recently, also from Invitrogen

78. Why HEK.EBNA Cells? Advantages In comparison to other eukaryotic expression systems the HEK.EBNA Expression System is rapid: from gene to protein in 4-6 weeks It can be applied to generate stable cell lines (pools/ clones) and in transient mode on small and large scale The cells can be grown adherently and in serum-free suspension culture In transient mode not only secreted and membrane- bound, but also intracellular proteins can successfully be expressed

79. HEK.EBNA Expression Vectors Basic vector (also Gateway™ adapted) Can be decorated with N- or C-terminal tags, heterologous leader sequences Co-expression of e.g. GFP via IRES element Selectable marker for generation of stable cell line Commercially available HEK.EBNA vectors: pREP4 and pCEP4 (Invitrogen)

80. A Transient Transfection Run….. 0 5 10 15 20 25 020406080100120140160180 time [h] cell density [ x 10 5 cells/ml] 0 1 2 3 4 5 6 7 8 9 10 product titer [mg/l] cell densityproduct titer Cell density in 3.6 volume prior to transfection Cell density after addition of 1.4 l transfection mix Cell density after addition of 5 l growth medium

81. ….in Multiparallel Fashion

82. Cell/Supernatant Harvest and Cell Lysis Cell concentrate Super natant Wave bag Secreted product in supernatant or Cell concentration Cell debris Clear Lysate Intracellular product: Cell concentrate + Lysis buffer Released product in cleared lysate Wave bag