1 Recombinant proteins in insect and mammal cells , F E B R U A R Y 2 3 RD

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Wilfrid Mosley
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1 1 Recombinant proteins in insect and mammal cells , F E B R U A R Y 2 3 RD

2 Bacteria Yeast Insect cells Mammalian Cell growth Medium complexity Rapid (30 min) Rapid (90 min) Slow (18-24h) Slow (24h) No No Yes Yes Cost Low Low High High Expression High Low-high Low-high Mow-medium 2

Post-translational modifications (PTMs) N/O-Glycosylation attaches a sugar ; one of the most important modification with significant effects on protein folding, conformation, distribution, stability

3 Post-translational modifications (PTMs) N/O-Glycosylation attaches a sugar ; one of the most important modification with significant effects on protein folding, conformation, distribution, stability and activity Phosporylation adds a phosphate to Ser, Thr, Tyr; critical in the regulation of cell cycle, growth, signal transduction Methylation adds a methyl group at Lys or Arg; increases hydrophobicity of proteins N-acetylation adds acetyl group to N-term or at Lys; initially found to be important for the regulation of histone activity in the nucleus, it also occurs on cytosolic proteins. S-nitrosylation: stabilization of proteins γ-carboxylation converts glutamate to γ-carboxyglutamate; facilitates calcium binding, which is important/essential for the function of the protein 3

4 Post-translational modifications Protein folding N-linked glycosylation O-linked glycosylation Bacteria Yeast Insect Mammals Not reliable None Usually reliable High mannose Very reliable Simple, no sialic acid Very reliable Complex N Y Y Y Phosphorylation N Y Y Y Acetylation N Y Y Y Acylation N Y Y Y g-carboxylation N N N Y 4

5 Post-translational modifications Protein folding N-linked glycosylation O-linked glycosylation Bacteria Yeast Insect Mammals Not reliable None Usually reliable High mannose Very reliable Simple, no sialic acid Very reliable Complex N Y Y Y Phosphorylation N Y Y Y Acetylation N Y Y Y Acylation N Y Y Y g-carboxylation N N N Y 5

6 Post-translational modifications Protein folding N-linked glycosylation O-linked glycosylation Bacteria Yeast Insect Mammals Not reliable None Usually reliable High mannose Very reliable Simple, no sialic acid Very reliable Complex N Y Y Y Phosphorylation N Y Y Y Acetylation N Y Y Y Acylation N Y Y Y g-carboxylation N N N Y 6

7 Baculovirus-insect cell expression systems 7

8 Baculovirus 8 16 th century: wilting disease of silk-producing larvae More than 600 species of insects: Lepidoptera (butterflies and moths) Hymenoptera (sawflies and wasps) Diptera (flies) Coleoptera (beetles) Since 1940s: biopesticides in crop fields Non-infected larvae of Mamestra brassicae Larvae of Mamestra brassicae 7 days after infection

9 Baculovirus Since 1990s: production of complex eukaryotic proteins in insect cell cultures 1- research purposes - no need of codon optimization - post-translational modifications: glycosylation, phosporylation, S-nitrosylation, methylation, N-acetylation Brickey et al Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system. Biochem Biophys Res Com, 173: Ardisson-Araujo et al A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification. Virol J. 10: human and veterinary purposes - vaccine against foot-and-mouth disease (FMD) of goat, sheep and other cloven-hoof animals (Li et al Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine. PlosONe, 3(5): e2283) - vaccine for prevention of H5N1 Avian influenza in chickens (Nwe et al Expression of hemagglutinin protein from the avion influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture. BMC Microbiol, 6:16) - production of the human nerve growth factor, rhngf (Nguyen et al Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhngf) in the baculovirus expression system. J Biotech, 31: ) - gene therapy (Kohlbrenner et al Successful production of pseudotyped raav vectors using a modified baculovirus expression system. Mol Terap, 12: )

10 Baculovirus Since 1990s: production of complex eukaryotic proteins in insect cell cultures 1- research purposes - no need of codon optimization - post-translational modifications: glycosylation, phosporylation, S-nitrosylation, methylation, N-acetylation (Brickey et al Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system. Biochem Biophys Res Com, 173: ) 2- human and veterinary purposes 10 - vaccine against foot-and-mouth disease (FMD) of goat, sheep and other cloven-hoof animals (Li et al Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine. PlosONe, 3(5): e2283) - vaccine for prevention of H5N1 Avian influenza in chickens (Nwe et al Expression of hemagglutinin protein from the avion influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture. BMC Microbiol, 6:16) - production of the human nerve growth factor, rhngf (Nguyen et al Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhngf) in the baculovirus expression system. J Biotech, 31: ) - gene therapy (Kohlbrenner et al Successful production of pseudotyped raav vectors using a modified baculovirus expression system. Mol Terap, 12: )

involved in crucial functions Two baculoviruses widely used in biotechnology: - Autographa california multiple

11 Baculovirus Insect pathogen with > 500 baculovirus isolated Double-strand circular supercoiled DNA virus in a rod-shaped capsid kbp size 53 baculovirus isolates completely sequenced (January 2011) High conservation of core genes involved in crucial functions Two baculoviruses widely used in biotechnology: - Autographa california multiple nulceopolyhedrovirus (AcMNPV) - Bombyx mori (BmNPV) Two different virion types: - budded virions (BV) - occlusion derived virus (ODV) 11

12 Baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) 12 Autographa californica (alfalfa looper - Lepidoptera) Polyhedrin: major constituent of the polyhedron matrix viral protein of 29 kda protection solubilized only in highly alkalin conditions

13 Baculovirus 13 Life cycle in 2 phases: 1- budded-virus to spread infection from cell to cell within individual 2- Occlusion-derived virus as dispersal agent between individuals and survival mechanism

14 Baculovirus 14 Schematic representation of the four phases of baculovirus gene expression in vitro

Baculovirus Life cycle in vitro recombinant virus by homologous recombination 15

the foreign gene into a transfer vector which contains flanking sequences which are

Homologous recombination takes place inside the insect cells, leading to recombinant

15 Baculovirus Life cycle in vitro recombinant virus by homologous recombination 15 Baculovirus genome is too large to directly insert foreign genes easily Cloning of the foreign gene into a transfer vector which contains flanking sequences which are homologous to baculovirus genome. Co-transfection into insect cells. Homologous recombination takes place inside the insect cells, leading to recombinant baculovirus which will produce the foreign protein Screening by plaque assay : occlusion-negative phenotype.

16 Commercially available from Pharmingen: - BaculoGold baculovirus DNA bears a lethal deletion and does not code for viable virus. The co-transfection with a complementing baculovirus transfer vector rescues the lethal deletion. Recombination frequencies exceed 99%. - Linearized AcRP23.lacZ baculovirus DNA is a modified AcNPV baculovirus DNA in which the polyhedrin was replaced by a lacz gene. The recombination disrupts the lacz gene, allowing a blue/white screening. 30% of virus will be recombinant - Linearized AcUW1.lacZ baculovirus DNA: same as precedent but lacz gene under the control of p10 promoter 16

17 Bac-to-Bac Invitrogen/LifeTech 17 in vitro-recombinant virus by site-specific transposition: use site-specific transposition with Tn7 to insert foreign genes into bacmid (baculovirus shuttle vector) propagated in E.coli

18 Bac-to-Bac BES - ThermoFisher 18 Gene under the control of polyhedrin promoter: high yield pfastbac1: virus which express unfused recombinant proteins pfastbac HT series allows expression of polyhistidine-tagged proteins which can be purified rapidly on metal affinity resins pfastbac DUAL vector contains two promoters (polyhedrin and p10) allowing expression of 2 genes pfastbac HBM TOPO vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal

19 FlashBAC Oxford Expression Technologies 19 combines the advantages of bacmid technology with homologous recombination in insect cells

20 FlashBAC Oxford Expression Technologies 20

21 FlashBAC Oxford Expression Technologies 21 flashbac Backbone virus DNA has a chia deletion, which prevents production of virus chitinase. This enzyme blocks the secretory pathway and its absence helps improve membrane and secreted protein production1. flashbac GOLD Backbone virus DNA has gene deletions for chia and v-cath. This avoids production of chitinase and cathepsin, a viral protease that may otherwise degrade susceptible target proteins. flashbac ULTRA Backbone virus DNA has deletions of chia, v-cath and p10/p26/p74. Deletion of p10 results in delayed cell lysis (particularly noticeable) in TnHi5 cells and thus can extend protein production times. It also reduces the metabolic burden on the cell of producing high levels of P10 protein. flashbac PRIME No gene deletions in the virus back bone. Useful if the proteins being expressed form complexes inside the cytoplasm or nucleus that need to be purified. We find that the relatively early cell lysis associated with PRIME makes it easier to purify these complexes e.g. VLPs.

22 Comparison of different systems 22

23 Insect cell lines 23

24 Insect cell lines 24

4 Aeration-Requires passive 02 diffusion for optimal growth & recombinant protein expression

25 Growing insect cells Monolayer culture Suspension culture 25 Requirements for proper insect cell culture: Temperature- Optimal range is C ph- Optimal range is 6.1 to 6.4 Aeration-Requires passive 02 diffusion for optimal growth & recombinant protein expression Osmolality- Optimum is mosm/kg Fetal Bovine Serum (FBS): source of nutrients and protection from cellular shear forces

26 Insect cell transfection Calcium phosphate coprecipitation: the method involves the formation of a coprecipitate of calcium phosphate and DNA that is taken up by the cells. DEAE-dextran-mediated transfection: DEAE-dextran is a polycation that binds both DNA and the cell surface, promoting thus adsoprtion of the DNA by the cell. Liposome-mediated transfection: this method employs the synthetic lipid DOTMA which was specifically designed to form positively charged liposomes that spontaneously interact with DNA. Upon addition to the cells, the liposome/dna complex binds to the ngatively charged cell surface. The liposome then fused with the cell membrane, efficiently introducing the DNA into the cells. Electroporation Cellfectin/Cellfectin II and more 26

27 Limitations of BV-insect cell expression systems BV infection ultimately results in cell death and lysis in a few days due to the very strong expression driven by Polh or p10 promoter Glycosylation in insect cells differs in many aspect from in mammalian cells: oligosaccharides are shorter in insect cells nature of N- and O-glucoside is different: Mammalian cells N-glucosides Complex (mannose, galactose, N- acetylglucosamine, neuramic acid) with sialylation O-glucosides 27 Galactose, N-acetylglucosamine, N-acetylgalactosamine, neuramic acid Insect cells Simple (mannose) but no sialylation Not identical but very similar to mammalian cells absence of sialylation of N-glycans in insect cells: problem for human therapeutics insect cells are not able to properly process proteins that are initially synthesized as large inactive precursor proteins

28 Protein expression in mammalian cells 28

29 Baculovirus gene transfer into Mammalian cells 29 Baculovirus can enter mammalian cells via endocytosis BUT they are not able to replicate into mammalian cells and require the insertion of a strong mammalian gene regulatory element

30 Baculovirus gene transfer into Mammalian cells 30 Known as BacMam and initiated by Boyce and Bucher Baculoviral transfer plasmid: lacz reporter gene under control of RSV (Rous sarcoma virus) promoter together with mammalian SV40 splice and polyadenylation signals

31 Baculovirus gene transfer into Mammalian cells 31 Transfer plasmid AcMNPV Recombination by co-transfection of Sf21 insect cells Purification of modified-acmnpv Transfection of a variety of mammalian cell lines

Mammalian cells 32 Expression cassette: - strong viral promoters from cytomegalovirus (CMV) or SV40 - Kozak sequence (ggrccaugg in higher vertebrate) - signal sequence for secretion - sequence tag -

32 Mammalian cells 32 Expression cassette: - strong viral promoters from cytomegalovirus (CMV) or SV40 - Kozak sequence (ggrccaugg in higher vertebrate) - signal sequence for secretion - sequence tag - cleavage site - poly-adenylation signal Expression vector: - antibiotic resistance gene for selection of stable mammalian cell line - antibiotic resistance gene for selection in E. coli - puc origin for multiplication in E. coli - multiple cloning site

33 Mammalian cells 33

34 Mammalian cells 34 Transfection of mammalian cells: - calcium phosphate (Chen and Okayama, 1987;Wigler et al., 1977), - lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) - and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988)

35 Mammalian cells 35 Cell line CHO DG44 DUK-B11 NSO HEK293 BHK COS PER.C6 Origin An epithelial cell line derived from the ovaries of Chinese hamsters A CHO cell line (marker: DHFR) A CHO cell line (marker: GS) A myeloma cell line derived from B lymphocytes of mice An epithelial cell line derived from human embryonic kidney cells transformed with adenovirus DNA A cell line derived from the kidney cells of baby Syrian golden hamsters Fibroblast cell lines derived from the kidney cells (SV40 transformed) of African green minkeys A trademarked cell line derived from a human retinal cell (fron Crucell Holland BV)

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37 Baculovirus in plants 37

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40 baculovirus vector can be used to express recombinant proteins in plants. baculovirus vector- mediated gene delivery and expression system could be used in plant biotechnology for fast and efficient production of recombinant proteins and for molecular virology studies in plants. 40

Oxford Expression Technologies Ltd

Oxford Expression Technologies Ltd Founded in 2007 as a spin out from Oxford Brookes University and Natural Environment Research Council Technology based on the insect baculovirus expression vectors (BEVs)