Expression insect cells

Incubations with Cloned, Expressed Enzymes Individual UGT enzymes have been expressed in a wide variety of systems including insect cells (Supersomes or Baculosomes), Escherichia coli, yeast, and mammalian cells. Zakim and Dannenberg have demonstrated that the lipid membrane composition can influence activity (Zakim, 1992). There tends to be excellent protein expression insect cells transfected with baculovirus, but when activity is measured compared to mammalian cells systems, there appears to be significant amounts of inactive protein due to either poor membrane insertion or improper folding (lack of chaperones ). Bacteria do not have an ER, but alteration of the signal sequence results in active membrane bound preparations. Yeast and mammalian cells such as HEK293 or V79 cells have a more typical membrane environment and may be preferable for expression of ER proteins. 

Tomita, M., Yoshizato, K., Nagata, K., and Kitajima, T. 1999. Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells. /Biochem, 126 1118-26. 

Subunit vaccines based on the surface proteins of vims are also being explored. It has been demonstrated that the two major protective antigens are haemagglutinin (HA) and neuraminidase (NA). The genes for these antigens have been cloned and expressed in baculovims in insect cell culture (84). 

The recent explosion in the discovery of new myosin genes has led to the idea that myosins from different classes probably co-exist in cells. This has raised the obvious question as to what functions these myosins subserve within cells. Up to now, only the genes have been cloned for many of the 35 unique myosins. But this is not a question that can be answered solely by cloning rather, it is absolutely imperative to biochemically characterize these proteins if we are to understand their physiological properties. One way to do this is to express the entire protein or parts of the proteins in bacteria, yeast, or insect cells, and to then purify and characterize... 

A number of allergens from both honey bee and vespid venoms have been cloned and expressed by either Escherichia coli or baculovirus-infected insect cells (table 1) phospholipase Aj [20], hyaluronidase [21], acid phosphatase [13] and Api m6 [14] from honey bee venom, as well as antigen 5 [22], phospholipase A and hyaluronidase [23] from vespid venom, and dipeptidylpeptidases from both bee and Vespula venoms [15, 16]. Their reactivity with human-specific IgE antibodies to the respective allergens has been documented [11-16, 22, 23] and their specificity is superior... 

Grunwald T, Bockisch B, Spillner E, Ring J, Brede-horst R, Ollert M Molecular cloning and expression and expression in insect cells of honey bee venom allergen acid phosphatase (Api m3). J Allergy Clin Immunol 2006 117 848-854. 

Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). 

Andersen, J.N., "Temperature Effect on Recombinant Protein Production Using a Baculovirus/Insect Cell Expression System", Diploma Thesis, University of Calgary and Technical University of Denmark, 1995. 

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

Also, special vectors allowing expression in both insect cells and mammalian cell cultures from the same vector (pMamaBac [11] andpBacMam [12]) were described, though the amount required for mammalian transfection with one of these vectors is twofold higher than for insect cells, which makes it applicable only for assessment of suitability for a certain cell culture. 

A plasmid-based transient expression system (InsectDirect system from EMD Biosciences Inc., USA www.emdbiosciences.com) will most probably greatly facilitate parallelization and automation for insect cell cultures. It generally gives lower yields, since expression is driven by an early baculoviral promoter, but it is possible to evaluate protein activity and expression level 24 h after transfection. It is also scalable to 1 L volume. The two main disadvantages, namely the large amount of transfection agent required and the limitation in scalability, can probably be overcome in future. 

Zhang, F., Saarinen, M.A., Itle, L.J. et al. (2002) The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system. Biotechnology and Bioengineering, 11 (2), 219-224. 

Huynh, C.Q. and Zieler, H. (1999) Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines. Journal of Molecular Biology, 288 (1), 13—20. 

Jarvis, D.L. (2003) Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology, 310 (1), 1-7. 

Co-expression of the human a- and p-subunits in the yeast Pichea pastoralis produces only trace amounts of active tetramer, with the majority being present in an unassembled form. Co-expression with human type III collagens, however, increases this assembly level tenfold. This indicates that collagen synthesis and the formation of an active prolyl 4-hydroxylase complex are mutually dependent processes (Vuorela et al, 1997). A similar observation has been noted for baculovirus encoded enzymes in insect cells (Lamberg et al, 1996). These findings support the hypothesis that this unusual control mechanism may be a common feature of collagen synthesis in all cell types. 

Although proteins can be expressed in many heterologous production systems, including bacteria such as Proteus mirabilis [1], fungi such as Pichia pastoris [2, 3] and Aspergillus awamori [4] and insect cells [5, 6], the pharmaceutical industry has narrowed down process development to a small number of platform technologies ... 

KUTCHAN, T.M., BOCK, A., DITTRICH, H., Heterologous expression of strictosidine synthase and berberine bridge enzyme in insect cell culture, Phytochemistry, 1994, 35, 353-360. 

Baculovirus Improved procedure Infection of insect cells High expression yields Relatively slow virus production Different post-translational processing... 

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