Expression in Eukaryotic Systems

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Expression in Eukaryotic Systems

• Yeast
• - Saccharomyces cerevisiae (bakers yeast)
• - Pichia pastoris
• Insect Cells Baculovirus
• Mammalian Cells

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Expression in Yeast
Autonomous replicating vectors -gt shuttle vectors
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Expression in Saccharomyces cerevisiaeAutonomous
replicating systems
Yep
Yrp
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Expression in Saccharomyces cerevisiaeIntegrative
systems
YIp
Probability for integration higher with linear
fragments !
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Yeast Artifical Chromosomes
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Yeast Artifical Chromosomes (YAC)
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Expression in Saccharomyces cerevisiae
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Yeast Promoter
UAS Upstream activating sequence URS Upstream
repressing sequence
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Expression in Saccharomyces cerevisiae
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Expression in Saccharomyces cerevisiae
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Yeast are efficient secretors ! Secretory
expression preferred if -gt if product toxic -gt
if many S-S bonds need to be closed
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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 -gt protein stack in
  cells (periplasma)
• S. cerevisiae produces high amount of EtOH -gt
  toxic for the cells -gt effects level of
  production
• Advantages of production in Pichia pastoris
• Highly efficient promoter, tightly regulated
  (alcohol oxidase -gt AOX, induced by MeOH)
• Produces no EtOH -gt very high cell density -gt
  secretion very efficient
• Secretes very few proteins -gt simplification of
  purification of secreted proteins

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Expression in Pichia pastorisIntegrative systems
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Expression in Pichia pastoris
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Expression in Pichia pastoris
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Expression in Pichia pastoris
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Mut -gt both AOX1 and AOX2 are functional -gt
methanol as inducer and as carbon source
used MutS -gt only AOX2 functional -gt methanol as
inducer and just to minder extend as carbon
sourse Mut- -gt non of the AOX are functional -gt
methanol only as inducer
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Knockout strains (Lab strain design)
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Glycosylation
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Expression in Pichia pastoris
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Yeast surface display
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Yeast Two-Hybrid System
-gt to study protein-protein interactions -gt
another possibility by SPR
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Expression in Insect cells

• Baculovirus
• -gt infects invertebrates (insects)
• -gt in infection cycle 2 forms of baculovirus are
  formed
• -gt
  single virus particle
• -gt
  in protein matrix (polyhedron) trapped clusters
  of viruses
• -gt during late stage of infection massive amount
  of polyhedron produced -gt strong promoter
• -gt polyhedron not required for virus production
• -gt polyhedron promoter optimal for heterologous
  protein production in insect cells

-gt Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV) often used as
expression vector
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Baculovirus expression system
-gt Production of recombinant baculovirus
1. create a transfer vector (E. coli based
plasmid with AcMNPV DNA polyhedrin
promoter/terminator flanking sequences)
-gt gene of interest cloned downstream
of promoter 2. Insect cells
are cotransfected with virus (AcMNPV)
transfer vector -gt in some double
infected cells -gt double
crossover event (recombination) -gt
produce recombinant virus (bacmid -gt
E. coli - insect cell baculovirus shuttle
vector) -gt cells infected
with recombinant virus -gt 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 -gt 4-5
days later protein harvested
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Baculovirus expression system
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Baculovirus expression system

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

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Drosophila cells as expression system
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Drosophila cells as expression system
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Drosophila cells as expression system
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Mammalian cell expression system

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

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Mammalian cell expression system

• 1. Adenoviral Vector
• 2. Cytomegalovirus (CMV) promoter
• 3. Inducible mammalian expression systems
• 4. Sindbis Virus

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Adenoviral expression system
Many cell types can be infected !!!
-gt used for gene therapy !!!
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Adenoviral expression system
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Adenoviral expression system
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Cytomegalovirus enhancer-containing promoter
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Cytomegalovirus enhancer-containing promoter
-gt positive and negative regulation of promoter
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Cytomegalovirus enhancer-containing promoter
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Inducible Mammalian Expression Systems
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Inducible Mammalian Expression Systems
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Inducible Mammalian Expression Systems
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Inducible Mammalian Expression Systems
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Inducible Mammalian Expression Systems
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Inducible Mammalian Expression Systems
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Expression using Sindbis Virus

• Broad host range
• High level expression

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Expression using Sindbis Virus

• Broad host range
• High level expression

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Expression using Sindbis Virus
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Mammalian cell expression system
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•  Gene expression in mammalian cell lines
• A convenient alternative for setting up mammalian
  cell facilities get a comprehensive service
  from us. We will achieve stable expression of the
  gene of your interest in mammalian cells.
• Customer provides
• - Mammalian vector with the gene (cDNA) to be
  expressed. We accept plasmid and retroviral
  vectors
• - Sequence of the gene and map of the construct
  for transfection
• Cell line or information about the cell line to
  be transfected.
• Our service includes
• - Transfection of the cells. In case of a
  retroviral vector, virus production and cell
  infection
• - Antibiotic selection and generation of stable
  transfected (infected) cell clones. At least 10
  independent clones will be selected and grown
• - Quantitative assay of the gene (cDNA)
  expression level in each transfected clone by RNA
  isolation followed by Northern hybridisation
  and/or RT-PCR
• - Selection of the best expressing clone
• - Cell freezing and depositing
• - Duration 3-6 months (depending on the cell
  growth rate), allow 1month in addition if the
  cell line is not available in our collections
• Customer receives