title: Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems.
abstract: The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.
authors.affiliation: Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL A1B 3V6, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: T I
authors.initials: TI
authors.lastname: Michalak
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005805:Genes, MHC Class I; D005821:Genetic Techniques; D005822:Genetic Vectors; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry; D007313:Insecta; D007371:Interferon-gamma / Q000737:chemistry* / Q000378:metabolism; D008285:Major Histocompatibility Complex; D008392:Marmota; D051379:Mice; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000998:Antiviral Agents; D004396:Coloring Agents; D016207:Cytokines; D018076:DNA, Complementary; D007074:Immunoglobulin G; D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C022616:thiazolyl blue