Insect cell

Insect Cells. In this system the cDNA is inserted into the genome of an insect vims, baculovims. Insect cells, or Hve insect larvae, are then infected with the vims. In this way advantage is taken of the vims s natural machinery for repHcation utilizing the insect cell. This is one of the best systems available for high level production of native protein having post-translational modifications similar to those seen in mammalian cells. Disadvantages of this system include lytic—batch variations, comparatively slow growth, and cosdy scale-up. 

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. 

Protein precipitate, insect cells Hvbridoma. Mammalian cells... 

Protein precipitate, hybridoma cells Insect cells Mammalian Cells... 

The uncertainty involved in the calculation of the energy dissipation rate makes it difficult to compare experimental results reported by different researchers. For the same reasons, so far it has proved difficult to assess flow induced effects in different items of process equipment using the common basis of equal energy dissipation rate. For example. Fig. 14 shows the biological response of (SF-9) insect cells as a function of the energy dissipation rates in a capillary tube and in a mechanically stirred vessel [99]. In these plots the calcula-... 

Fig. 14. Calcium response of Sf-9 insect cells subjected to different values of e in a stirred bioreactor equipped with a 5.1 cm diameter 6-bladed Rushton impeller (closed circles) or in the capillary flow system (open squares). Error bars for stirred bioreactor are standard deviation for each experiment but for the capillary, data are hard to discern [99]...

The shear forces are mainly in the range of 1 to lONm. This exposure causes cell death between 20 and 80% depending on the exposure duration which is between a few seconds and several hours. Studies performed in a bioreactor have an exposure duration of several days. The results are partly contradictory. Tramper et al. [30] found a critical stress level of 1.5 Nm" for insect cells, whereas Oh et al. [31] could not show an influence on hybridoma cells even at high stirrer speed. This shows that each cell line reacts different and that there is a necessity for defined stress systems if the results is to be comparable. 

Effect of Temperature on Insect Cell Growth Kinetics... 

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. 

E. coli B. subtilis Pseudomonas sp. Yeasts Insect cells Mammalian cells Plants ... 

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. 

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