Development of a baculovirus expression system for soluble porcine tumor necrosis factor receptor type I and soluble porcine tumor necrosis factor receptor type I-IgG fusion protein.
Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of inflammatory responses and gram-negative bacterial sepsis, but the role that it plays during Salmonella enterica species bacterial infections in swine has not yet been elucidated. To facilitate studies on the role of TNF-alpha on the pathology associated with Salmonella infections in pigs, recombinant soluble porcine TNF receptor type I (rspTNF-RI) and soluble TNF receptor type I fused to the Fc region of porcine IgG1 (rspTNF-RI-IgG) were expressed in insect cells using a baculovirus expression system. The proteins were secreted into the cell culture media and purified by anti-soluble porcine TNF-RI antibody and protein G affinity chromatography, respectively. The yield of protein using this method was approximately 1.5mg rspTNF-RI and 4mg rspTNF-RI-IgG/L of cell culture medium. In in vitro assays, rspTNF-RI-IgG was approximately 10-fold (0.97 vs. 10.00pmol/ml) more effective than rspTNF-RI at completely inhibiting the cytotoxic activity of 500U of recombinant porcine TNF-alpha on 3 x 10(4) WEHI 164 murine fibrosarcoma, clone 13, cells. Compared to previously described methods, this method yields significantly more biologically active rspTNF-RI.
Mwangi
Simon Musyoka
SM
National Animal Disease Center, ARS, USDA, 2300 Dayton Avenue, Ames, IA 50010, USA.
Stabel
Thomas J
TJ
Kehrli
Marcus E
ME
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000378:metabolism / Q000821:virology; D013552:Swine; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D015703:Antigens, CD; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha