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Expression in Eukaryotic Systems

Expression in Eukaryotic Systems. Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris Insect Cells – Baculovirus Mammalian Cells. Expression in Yeast. Autonomous replicating vectors -> shuttle vectors.

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Expression in Eukaryotic Systems

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  1. Expression in Eukaryotic Systems • Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris • Insect Cells – Baculovirus • Mammalian Cells

  2. Expression in Yeast Autonomous replicating vectors -> shuttle vectors

  3. Expression in Saccharomyces cerevisiaeAutonomous replicating systems Yep Yrp

  4. Expression in Saccharomyces cerevisiaeIntegrative systems YIp Probability for integration higher with linear fragments !

  5. Yeast Artifical Chromosomes

  6. Yeast Artifical Chromosomes (YAC)

  7. Expression in Saccharomyces cerevisiae

  8. Yeast Promoter UAS: Upstream activating sequence URS: Upstream repressing sequence

  9. Expression in Saccharomyces cerevisiae

  10. Expression in Saccharomyces cerevisiae

  11. Yeast are efficient secretors ! Secretory expression preferred if: -> if product toxic -> if many S-S bonds need to be closed

  12. Expression in S. cerevisiae – Pichia pastoris Problems with production in S. cerevisiae: • For some proteins production level low • Hyperglycosylation (more than 100 mannose residues in N-glycosylation) • Sometimes secretion not good -> protein stack in cells (periplasma) • S. cerevisiae produces high amount of EtOH -> toxic for the cells -> effects level of production Advantages of production in Pichia pastoris: • Highly efficient promoter, tightly regulated (alcohol oxidase -> AOX, induced by MeOH) • Produces no EtOH -> very high cell density -> secretion very efficient • Secretes very few proteins -> simplification of purification of secreted proteins

  13. Expression in Pichia pastorisIntegrative systems

  14. Expression in Pichia pastoris

  15. Expression in Pichia pastoris

  16. Expression in Pichia pastoris

  17. Mut+ -> both AOX1 and AOX2 are functional -> methanol as inducer and as carbon source used MutS -> only AOX2 functional -> methanol as inducer and just to minder extend as carbon sourse Mut- -> non of the AOX are functional -> methanol only as inducer

  18. ”Knockout” strains (Lab strain design)

  19. Glycosylation

  20. Expression in Pichia pastoris

  21. Yeast surface display

  22. Yeast Two-Hybrid System -> to study protein-protein interactions -> another possibility by SPR

  23. Expression in Insect cells • Baculovirus: -> infects invertebrates (insects) -> in infection cycle 2 forms of baculovirus are formed: -> single virus particle -> in protein matrix (polyhedron) trapped clusters of viruses -> during late stage of infection massive amount of polyhedron produced -> strong promoter -> polyhedron not required for virus production -> polyhedron promoter optimal for heterologous protein production in insect cells -> Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) often used as expression vector

  24. Baculovirus expression system -> Production of recombinant baculovirus: 1. create a transfer vector (E. coli based plasmid with AcMNPV DNA – polyhedrin promoter/terminator + flanking sequences) -> gene of interest cloned downstream of promoter 2. Insect cells are cotransfected with virus (AcMNPV) + transfer vector -> in some double infected cells -> double crossover event (recombination) -> produce recombinant virus (bacmid -> E. coli - insect cell baculovirus shuttle vector) -> cells infected with recombinant virus -> produce plaques (lack of polyhedrin) 3. DNA hydridisation + PCR used to identify recombinant virus 4. Infection of insect cells with concentrated stock of verified recombinant virus -> 4-5 days later protein harvested

  25. Baculovirus expression system

  26. Baculovirus expression system Why this system? • Insect cells have almost the same posttranslational modifications as mammalian cells • Higher expression level than mammalian cells

  27. Drosophila cells as expression system

  28. Drosophila cells as expression system

  29. Drosophila cells as expression system

  30. Mammalian cell expression system 1. Why do we use that system? -> to get full complement of posttranslational modifications on proteins 2. Developed cell lines: -> short term (transient) expression -> autonomous replicating systems -> viral origins (SV40) - African green monkey kidney (COS) - baby hamster kidney (BHK) - human embryonic kidney (HEK-239) -> long term (stable) expression -> integration into chromosome -> viral origins - chinese hamster ovary (CHO)

  31. Mammalian cell expression system 1. Adenoviral Vector 2. Cytomegalovirus (CMV) promoter 3. Inducible mammalian expression systems 4. Sindbis Virus

  32. Adenoviral expression system Many cell types can be infected !!! -> used for gene therapy !!!

  33. Adenoviral expression system

  34. Adenoviral expression system

  35. Cytomegalovirus enhancer-containing promoter

  36. Cytomegalovirus enhancer-containing promoter -> positive and negative regulation of promoter

  37. Cytomegalovirus enhancer-containing promoter

  38. Inducible Mammalian Expression Systems

  39. Inducible Mammalian Expression Systems

  40. Inducible Mammalian Expression Systems

  41. Inducible Mammalian Expression Systems

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