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-α) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was pla...

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Bibliographic Details
Published in:Veterinary immunology and immunopathology 2000-12, Vol.77 (3), p.233-241
Main Authors: Mwangi, Simon M, Stabel, Judy, Lee, Eun-Kyung, Kehrli, Marcus E, Taylor, Michael J
Format: Article
Language:English
Subjects:
Animals
Antigens, CD - biosynthesis
Antigens, CD - isolation & purification
Bovine
Cattle
Expression
Pichia
Pichia - genetics
Pichia pastoris
Polymerase Chain Reaction
Receptor
Receptors, Tumor Necrosis Factor - biosynthesis
Receptors, Tumor Necrosis Factor - isolation & purification
Receptors, Tumor Necrosis Factor, Type I
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Tumor necrosis factor alpha
tumor necrosis factor receptors
Tumor Necrosis Factor-alpha - antagonists & inhibitors
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Summary: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-α) 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-α on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 μg/ml, and by nearly 90% when used at a concentration of 310 μg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.
ISSN:0165-2427
1873-2534
DOI:10.1016/S0165-2427(00)00244-0