Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I.
A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.
Mwangi
S M
SM
Biomedical Sciences Department, College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA.
Stabel
J
J
Lee
E
E
Kehrli
M E
ME
Taylor
M J
MJ
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis* / Q000302:isolation & purification; D002417:Cattle; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000302:isolation & purification; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D015703:Antigens, CD; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha