Partial Purification and Characterization of Host Cell Proteases in the SF-9 Insect Cell Line
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The SF-9 cell line, like many other cell lines, is used by many industrial and academic laboratories for the expression of recombinant proteins. The SF-9 cell line was cloned by G.E. Smith and C.L. Cherry in 1983 from the parent line, IPLB-SF 21 AE, which was derived from pupal ovarian tissue of the fall army worm, Spodoptera frugiperda, by Vaughn, et aI., in 1977. The SF-9 cell line is highly susceptible to infection with baculoviruses, and can be used with baculovirus expression vectors for the expression of recombinant (genetically engineered) proteins. Laboratories, acknowledging the susceptibiIity of SF-9 cell line with baculoviruses, have used the cell line for the production of genetically engineered proteins. One major problem observed when the SF-9 cell line is used for the production of proteins is the proteolytic activity of the cell line, which hinders the production of any desired protein by reducing the yields, or by disrupting the composition of the final product. At the present time information concerning SF-9 protease activity, protease characteristics, or inhibitors is lacking. The purpose of this project is to partially purify and characterize the SF-9 proteases. For the isolation of the SF-9 proteases, various methods of affinity chromatography, ion exchange and size exclusion chromatography were used. For the characterization of the SF-9 proteases, the methods of SDS- PAGE and substrate specificity studies were performed, and inhibitor studies were performed using a variety of known protease inhibitors. Through development of an assay for the detection of protease activity using chromogenic substrates, a method was developed for the use of substrates as indication of the proteases inhibition. With the use of DEAE and SEC column, 3 proteases were partially purified and characterized. The proteases were identified as serine proteases with the use of class-specific protease inhibitors. Molecular weight of one of the proteases is approximately 59,000 d, while the other two proteases have the molecular weight of 42,600 d.