Sf9 cells

Akita, H., et al. Transport activity of human MRP3 expressed in Sf9 cells comparative studies with rat MRP3. Pharm. Res. 2002, 39, 34-41. 

Fabian, J. R., Kimball, S. R., Heinzinger, N. K., and Jefferson, L. S. (1997). Subunit assembly and guanine nucleotide exchange activity of eukaryotic initiation factor-2B expressed in Sf9 cells. J. Biol. Chem. 272, 12359—12365. 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

Sf9 cell culture at 1-2 x 10 cells/ml 1 pig linearized BaculoGold DNA (BD Blosclences) Recombinant Baculovirus transfer vector containing the Insert... 

Sf9 cell culture 150-mm tissue culture plates High-titre recombinant viral stock EX-CEL 405 serum-free medium containing 50 xg/ml gentamycin sulphate, 2.5 i.g/ml amphotericin B, and 10% fetal calf serum 27°C incubator Microscope Haemocytometer... 

Pipette Sf9 cell culture containing 2x10 cells/ml into a spinner flask. 

In Section 2, factors that could lead to particle assembly and secretion into the supernatant were discussed. At this point a deeper analysis of the factors affecting cell infection will be made. Optimisation of the production process should take into account virus-cell interactions, and more specifically viral attachment and internalisation into the cell. The impact of chemical modifications of the medium in baculovirus attachment-internalisation has not been carefully studied. It is widely known for example, that serum increases the infec-tivity of baculovirus. These reviewers have had one case where we were only able to succeed in infecting Sf9 cells adapted to growth in serum-free media [52], with a baculovirus produced by Sf9 cells (not adapted to grow in serum-free media), after adding serum to the culture (authors unpublished observations). However, since serum is not desirable for use in industrial production, its utilisation should be avoided as much as possible. 

The range of carbohydrate utilisation differs between different insect cell lines. Glucose, fructose and maltose can be used individually as carbon sources in Sf9 cell cultures [42]. 

It has been reported that specific oxygen uptake rate (OURsp) increases transiently after baculovirus infection (reviewed by Agathos [56]). This increase can vary between 7 and 100%. In the production of HIV-CLPs, Cruz et al. reported a 100 % increase in OURsp of Sf9 cells after baculovirus infection [69] although no increase was detected in PPV-VLP production (authors unpublished results). 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

Overall influence of DO levels in product expression and insect cell growth was reviewed by Agathos [56]. Generally there are indications that Sf9 cells show similar growth characteristics at 10 to 70% air saturation. This has been confirmed more recently by Cruz et al. [69] for Sf9 cells in DO levels in the... 

It is of interest that studies with cloned human cytochromes P450 expressed in insect SF9 cells have shown that only CYP2A6 catalysed 7-hydroxylation, while the formation of orl/20-hydroxyphenylacetaldehyde was supported by CYPlAl, 1A2, 2B6, 2E1 and 3A4 (Zhuo et al, 1999). 

RK is cloned and expressed in SF9 cells [17,23,32] cDNA encoding RK is characterized and sequenced, 561 amino acids protein [22] RK gene expression in baculovirus-infected Sf21 cells [25,30] RK gene is cloned and expressed in Pichia pastoris GS115, COS-1 cells and HEK-293 stable cell line, best in COS-1 cells with correct posttranslational modifications [28] expression of RK and mutants in COS-7 cells [33]) [17, 22, 23, 25, 28, 30, 32, 33] <4> (cDNA encoding enzyme is cloned and expressed in COS-7 cells, sequence of the 561 amino acids protein [13]) [13]... 

Glu-tagged PI 3-kinase G2 protein is expressed in both HEK293 cells and Sf9 cells [2]) [1, 2]... 

Hepler JR, Kozasa T, Smrcka AV, Simon MI, Rhee SG, Sternweis PC, Gilman AG (1993) Purification from Sf9 cells and characterization of recombinant Gq alpha and Gil alpha. Activation of purified phospholipase C isozymes by G alpha subunits. J Biol Chem 268(19) 14367-14375... 

Figler, R. A., Graber, S. G., Lindorfer, M. A., Yasuda, H., Linden, J., and Garrison, J. C. (1996). Reconstitution of recombinant bovine Ai adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition. Mol. Pharmacol. 50, 1587-1595. 

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