| 摘要 |
Non-carboxylated sulfated polyanions 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 the expression of recombinant protein, but which clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The three most effective polyanions for inducing a single-cell suspension were dextran sulfate, polyvinyl sulfate, and pentosan sulfate. The cost of dextran sulfate treatment is low compared to heparin treatment, which required a 20-fold higher lever to induce single-cell suspension. More importantly, dextran sulfate does not block vital infection at MOI>/=1 whereas heparin is known to seriously inhibit infection. To overcome this effect, the cells can be subcultured to a fresh culture medium without the sulfated polyanion, or the sulfated polyanion is neutralized with a polycation, then the cells are inoculated with the baculovirus. Once the cells are infected with the baculovirus, the sulfate polyanion is added back to the fresh culture medium. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in an increased volumetric yield of recombinant protein. Examples chosen for experimentation included, human secreted alkaline phosphatase, and b-galactosidase. Optimum volumetric yield of 6 to 11 fold higher than attached cells for the production of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up.
|
