Cloning and expression of porcine recombinant soluble tumor necrosis factor receptor 1.
OBJECTIVE
To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1).


METHODS
A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay.


RESULTS
A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 microg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner.


CONCLUSIONS
Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine.
Maroushek Boury
N
N
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames 50011, USA.
Bosworth
B T
BT
Stabel
T J
TJ
Kehrli
M E
ME
Taylor
M J
MJ
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004306:Dose-Response Relationship, Immunologic; D015870:Gene Expression; D015151:Immunoblotting / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012481:Salmonella Infections, Animal / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013552:Swine; D013553:Swine Diseases / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D015703:Antigens, CD; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha