| The insect cell-baculovirus expression system has been used extensively to produce recombinant proteins. Recently, a largely attachment-dependent cell line, Trichoplusia ni BTI-TN5B1-4, has been shown to be superior to commonly used insect cell lines for expression of both cytoplasmic and secreted glycosylated proteins. However, BTI-TN5B1-4 clumps severely in suspension with a concomitant drastic drop in per cell production, which has greatly limited the use of the cells for recombinant protein production.; Sulfated polyanions, such as dextran sulfate, pentosan sulfate and polyvinyl sulfate, have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for recombinant protein production but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. Inducing single-cell suspension resulted in a 2- to 4-fold increase in volumetric yields of model proteins and significantly higher per cell production compared to the commonly used Spodoptera frugiperda cell lines.; Sulfated polyanions, however, inhibited baculovirus infection of BTI-TN5B14 cells. Data from binding studies and fusion assays suggest that the inhibition of infection was not due to the observed reduction in viral attachment rate, but to inhibition of viral membrane fusion in the endosome. Simple infection strategies are proposed to mitigate the inhibition of viral infection, allowing the routine use of sulfated polyanions to maintain single-cell suspension of BTI-TN5B1-4 cells. Dextran sulfate is recommended for routine use because it is inexpensive, very effective, and does not significantly affect viral infection.; A quantitative understanding of virus-cell interaction would be useful in treating viral-mediated diseases, developing protocols for viral gene therapy, designing infection regimens for viral expressions systems, and optimizing vaccine and recombinant protein production. Here, a general mathematical model of the the trafficking of acid-dependent viruses that considers the various infection steps, such as binding, internalization, endosomal sorting, endosomal fusion, and nuclear accumulation (for DNA virus), is presented. The model mimics accurately the trafficking dynamics of baculovirus and Semliki Forest virus. Simple concepts from the model were then used to methodically design synchronous infection regimens for various insect cell lines, analyze the inherent inefficiency of existing assays for baculovirus titer, and develop an optimized assay. |
