Insect cells expression systems

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Insect cells expression systems
Pilailuk Akkapaiboon
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Why express recombinant protein?
Purpose Yield Required Study
protein function ug Generate
antibodies mg Analyze protein structure
100s mg Tool for diagnosis or therapy mg
gm Industrial/Military uses
kg train car load
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Gene Expression Systems
Highest Quality and Function
Easy-to-use
Mammalian
Prokaryotic
Insect
yeast
Lowest Cost
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Problems With Expression of Eukaryotic Genes In
Bacteria
1. Protein instability (proteolysis) 2. Presence
of contaminants from bacterial cell membranes in
the final product 3. Authentic eukaryotic protein
not produced Bacteria may not perform
posttranslational modifications of eukaryotic
proteins -Disulfide bond formation for proper
protein folding -Proteolytic processing of
precursor proteins -Reactions that specifically
add chemical groups to certain amino
acids Acetylation acetate Acylation fatty
acids Myristylation addition of 14 carbon
chain to N-terminal amino acid Palmitoylation
addition of 16 carbon chain to sulfhydryl group
of cysteine Carboxylation -COOH
Glycosylation, O- and N-linked carbohydrates
Hydroxylation -OH Phosphorylation
-PO4 Sulfation -SO4   Functions -Protein
structure and stability -Enzyme
activity -Anchoring to membranes -Secretion
from cell
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Recombinant Protein Production In Eukaryotic
Cells

• Yeast Expression Systems
• II. Insect Cell Expression Systems
• III. Mammalian Cell Expression Vectors

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Baculovirus Expression Vector System

• most powerful and versatile
• used to express heterologous genes from many
  different sources, including fungi, plants,
  bacteria and viruses in insect cells

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Baculovirus life cycle
Autographa californica multiple
nucleopolyhedrovirus (AcMNPV) 1. a budded virus
(BV) cell-to-cell transmission in the early
stage of infection 2. an occluded virus (OB)
protect the virus and allow to survive between
hosts, within the environment
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Generating a recombinant Baculovirus
Autographa californica multiple
nucleopolyhedrovirus (AcMNPV)

• Transfer vector contains
• an E.coli origin of replication
• an apicillin resistance marker
• a promoter from the polyhedrin
• a cloning region downstream from promoter
• a large tract of AcMNPV sequence flanking the
  cloning region

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Expression in Baculovirus system
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Baculovirus Expression Vector System

• high expression levels
• easy and fast scale-up to volume production
• limitless size of the expressed protein
• efficient cleavage of signal peptides and
  processing of the protein
• supports post-translational modifications
• simultaneous expression of multiple genes
• expressed proteins are correctly folded and
  biologically active
• safety

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Insect cell lines
Ovarian tissues of Spodoptera frugiperda Sf9,
Sf21 Doubling time 18-24 h Growth
temperature 27C CO2 supplementation
not required
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Spodoptera frugiperda (Sf9)
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Cytopathic effect of Spodoptera frugiperda (Sf9)
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Plaque assay
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Baculovirus Amplification
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Critical issues for gene expression projects
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Gene expression issues and solutions
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Gene expression and host comparison
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Develop a strategy that fits your protein