Production of antistasin using the baculovirus expression system
1991
Jain, D. | Irāmacupramaṇiyan̲, Ka | Gould, S. | Seamans, C. | Wang, S. | Lenny, A. | Silberklang, M.
We have used Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus to produce antistasin, a potent anticoagulant and antimetastatic agent from leech salivary glands. Mature antistasin is a 119-a.a.,15-kDa secreted protein which has an unusually high cysteine content (20/119 residues) and acts by inhibiting coagulation Factor Xa. We have also genetically engineered a truncated (7.5 kDa) form of antistasin, "half antistasin" (H-ANS) for baculovirus-mediated expression. The efficiency of utilization of baculovirus in this system was improved by the use of a low (0.1) multiplicity of infection. By monitoring increase in cell diameter during the course of infection and decrease in cell viability, both of which were found to correlate with product accumulation, harvest time could be optimized under various culture conditions. Secreted H-ANS activity peaked between 72 and 96 hours post-infection. Among the major process variables examined, dissolved oxygen at a low level of 10% and at a high level of 110% air saturation was found to result in a reduced growth rate as well as lower productivity relative to a level of 65%. Data are presented on the kinetics of oxygen uptake during cell growth and viral infection cycle for the production of H-ANS
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