Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9 cell cultures.

This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII. Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection. The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection. In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique. Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase.

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth.

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special((R)) enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N((R)) enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.