Non-obligatory/Obligatory Transient/stable

1. Strategies Non-obligatory/Obligatory Transient/stable Yes No Produce (and purify) the proteins independently and reconstitute the complex in vitro Co-expression is mandatory Co-expression in E. coli or in insect cells Purification of Endogenous complexes • Single gene expression • E. coli • Insect cells baculovirus • Yeast • Mammalian

2. Identification, production and purification Identification methods Purification strategies Single gene expression and in vitro assembly Co-expression techniques

3. Recombinant protein production Prokaryotic E. coli, Anabaena .... Eukaryotic Yeast Insect cells Mammalian Cell free systems: E coli Wheat germ, Insect

4. Recombinant protein production in E. coli Strain: XL1-Blue, BL21, …. Expression vector antibiotic resistance replication origin promoter

5. Lac Promoter

6. pBAD vectors

7. pET vectors BL21 [DE3]

8. pET vectors T7 promoter ColE1

9. Native and tagged proteins

10. Yeast expression PCR, cloning, transformation S. cerevisiae P. pastoris Copper inducible Methanol inducible Replicative Integrative Intracellular Secretion vectors Different tag systems Streamlining of workflow by recombinational in vivo cloning in S. cerevisiae

11. EBNA-1 protein drives episomal replication ofori-P containing plasmids integrated Ad5 E1a/E1b fragment in HEK 293 cells enhances trans- cription of CMV promotor driven transgene Why HEK.EBNA Cells? The Principle 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

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

13. HEK.EBNA Expression Vectors • Basic vector (alsoGateway™ adapted) • Can be decorated withN- or C-terminal tags, heterologous leadersequences • 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)

14. 25 10 Cell density in 3.6 volume prior to transfection 9 Cell density after addition of 1.4 l transfection mix 20 8 cells/ml] Cell density after addition 7 of 5 l growth medium E. Coli DH5 5 15 6 product titer [mg/l] 5 cell density [ x 10 10 4 3 5 2 1 0 0 0 20 40 60 80 100 120 140 160 180 time [h] cell density product titer Transient transfection Vector purification 20 l fermentation 30 g cell paste 15-30 mg plasmid

15. For Secreted Proteins: affinity chromatography on antibody or Protein A column -tag dependent- For Intracellularly Expressed Proteins: his and/or his-Strep tag  Ni-chelate and/or Streptactin column (oxford)

16. The BaculoVirus Expression System (BEVS)

17. What are baculoviruses ? Baculoviruses are a group of viruses found mostly on insects They are rod-shaped (latin baculum = stick), 40-50 nm in diameter and 200-400 nm in lengh Double stranded , covalently closed and circular DNA (80 – 200 kbp) Trichoplusia ni Spodoptera frugiperda

18. In cell culture or when multiplying within an insect host, baculoviruses Form so called virions, also referred to as non-occuded or budded virus (BV) For long-term survival occlusion bodies (OB) or polyhedra are formed. Para-crystalin matrix, composed of polyhedrin (50% of the total protein mass)

20. Baculovirus Life Cycle Early phase (0-6 post infection) • Enters the cells by endocytosis and the nucleocapsid rapidly migrate to nucleus • Viral DNA is realeased inside the nucleus and early gene expression starts Late phase (6-20 post infection) • Extensive DNA replication and subsequent production of budded virus particles after 12 p.i. • Projeny nucleocapsids leave nucleaus and axquire an envelope as the leav ethe cytoplasm as budded virus • 36-48h post infection • polyhedrin protein traps many virions into polyhedron “package” • polyhedron is stable Very Late phase (after 20 hrs post infection) • Decrease in the formation of budded virus (BV) • Nucleocapsids acquire an envelope inside the nucleus to form multiple nuclear polyhedrosis virus (MNPVs) • MNPVs are embodied in the polyhedrin matrix and fromation of occlusion bodies • Accumulation of the p10 protein in fibrous material • Cell lysis begins 60_72 hours post infection

21. Baculovirus Polyhedrin Promoter • biotechnologists have utilized this system • polyhedrin promoter is strong • don’t need polyhedron “package” in lab • replace polyhedrin coding seq. with GOI • Express lots of protein 36-48 h post-infection • Protein production in a eukaryotic host • Proper folding • Post-translational modifications

22. Baculovirus System • Virus utilized • AcMNPV • Autographa californica multiple nuclear polyhedrosis virus • A. californica = alfalfa looper • AcMNPV infects 30+ insects • Commonly used cell line • Fall armyworm - Spodoptera frugiperda (Sf9) • Polyhedrin promoter very active

23. Post-Translational Modifications • Possible modifications Insect cells expresssing heterologous proteins in the BVES appear to be able to cope with post-translational modifications: Signal peptide cleavage, Phosphorylation, Glycosylation, Acetylation, sulfation, disulfide bond formation... • Limitations Kind or degree of modification may not always be identical to the ones found in the original species or tissue backgound of the gene For structural biology post translational modification and especially glycosylation are problematic (source of heterogeneity)...

24. Disadvantages of Baculovirus Expression System • Insects cells have a large doubling time • Baculovirus system kills the cells - not continuous production • Some proteins not modified correctly • Insect cell culture is expensive and time consuming

25. Safety considerations • Personal risk or contamination of mammalian cells - baculovirus can enter mammalian cells but not reach nuclei or start gene expresion - baculoviruses are considered to pose no additional safety risk and are classified as Class I (like E. Coli K12) • Contaminating other insect cells - although their host range is fairly limited, baculoviruses are able to enter other other insect cells but not to replicate their DNA or enter late infection stages - care must be taken not to contaminate other insect work (baculoviruses can be used as pesticides) Like other recombinant organisms, baculoviruses are potential biohazards and care must be taken regarding their distribution and containment

26. Flowchart for Baculovirus Expression Clone the gene(s) of interest into a bacterial transfer vector Generate the recombinant virus Transfection/Co-transfection Small scale expression assay Prepare a high titer virus stock <108 pfu/ml Optimization of the expression conditions and large scale production

27. Basic concepts • Several cell lines (Sf9, Sf21, H5) and media (TNMFH, SF900-II, Express five...) are availible • Cells can be grown on monolayers (T25, T175) or in suspension (Deep Well, Spinner Flask, Erlenmeyer, Wave bag, Bioreactor) • Pfu: plaque forming unit Pfu/ml is the measure of viral titre, equivalent to the concentration of viruses • MOI: multiplicity of infection Number of viruses per cell. Usually low for virus amplification (MOI<0.1), hight for protein production (1<MOI)

28. What is needed to express a protein ? The expression unit • Strong promoter: p10 or PH • Kozak sequence: • Gene of interest • Terminator On both sides, elements that will allow the integration of the expression unit(s) into the viral genome • Segment from viral genome for homologous recombination in insect cells between the transfert vector and the viral DNA • Transposons (Tn7L and Tn7R) recombination sites (LoxP) when a bacmid is to be used

29. Homologous recombination The transfer vector and the viral DNA are co-transfected

30. A transfer vector

31. Transposition in E. Coli

32. What is needed ? • The target gene cloned into the transfer plasmid • donor plasmid • Bacmid formed by integration of E. coli plasmid into AcMNPV genome • double crossover event • ori of replication, select. marker, etc. • 3rd plasmid provides proteins to move gene of interest into bacmid • Helper plasmid

33. What is a bacmid ? AcMNPV genome E.coli plasmid Bacmid

34. Recombinant Bacmid Bacmid lacZ will be disrupted - white colonies on X-Gal Helper Plasmid with transposition genes Donor Plasmid Recombinant Bacmid

35. A donnor plasmid

36. Single gene baculovirus transfer vectors Numerous transfer vectors adapted are availible - Recombination in E. coli or in insect cells - Ligation based or Gateway based cloning - Expression of Native, N- or C- terminal tagged proteins GST Native Flag Hemagglutinin His6 His6 CBP … … Gateway Kozak ATG pBacGW F. Klein, S. Schochat, Y. Trottier, A. Poterszman, L. Salim, D. Busso Multi-system: TriEx or Gateway

37. Flowchart for Baculovirus Expression Clone the gene(s) of interest into a bacterial transfer vector Generate the recombinant virus Transfection/Co-transfection Small scale expression assay Prepare a high titer virus stock <108 pfu/ml Optimization of the expression conditions and large scale production

38. Homologous recombination in insect cells (FlashBac, Pharmingen, tri-ex) Homologous Recombinaison in E.Coli (Bacmids)

39. Day -5 Bacmids Cloned cDNA transformation in E. coli DH10bac/Multibac selection of recombinant bacmids (white colonies) bacmid preparation (Mini-prep) Transfer vectors co-transfection of the transfer vector with the viral genome in insect cells transfection of the bacmid in insect cells Day 0 Day 0 Day 7-10 Initial virus stock P0, # 4 ml 7 Days/round Amplification, P1, P2 purification, titration Small scale analysis (6 Well plate) Day 12-15 Optimization Large scale production

40. Upscaling and purification issues High titer virus stock Sf9, Hi5, Tni cells in exp phase T = 0 h Infection T= 48 or 72 Harvest Optimization of the expression conditions and large scale production

41. Titering a virus stock TidiousInfected cells do not divide - Plaque assay - end point dilution - rt pcr - viability assays

42. Optimization For infections cells in exponential growth phase are required. infect cells a 0.5 to 2.0 1O6 cell/ml T =27 °C Monolayers or suspension (Deep Well, Spinner, Bottles..) Optimization fo the culture conditions - harvest time post-infection: 48, 72, 96 hrs - multiplicities of infection: 0.1, 1, 5, 10Very important for co-infections experiments - cell line/media of choice: Sf9, Sf21, H5 with or without serum Small scale experiments: 4 ml cultures

43. Towards simplification and automation Culture Lysis Centrifugation 24-Deep well block (3ml culture) Purification Plaque 96-wells (batch purification) Coomassie Analysis

45. Nucleic acid synthesis Chemical synthesis: DNA or RNA oligonucleotides (up to 40 bps) Introduction of modified bases In vitro synthesis: DNA :DNA polymerase by PCR (100-500 bp for em) RNA: T7 RNA Pol In vivo synthesis: tRNAs, DNA (100-500 bp for em)

46. Production : par transcriptionin vitro / PolT7 linéarisation pUCT7 polT7 NTPs Matrices plasmidiques RNA transcrit linéarisation pRZ/pHDV polT7 NTPs + Mg RNA d’intérêt (3’ franc) + ribozyme Ou oligos polT7 NTPs RNA court transcrit Anne Catherine Dock-Bregreon