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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Publications at this Location » Publication #74654
Title: CLONING AND EXPRESSION OF THE EXTRACELLULAR DOMAIN OF PORCINE TNF RECEPTOR I

Author
item MAROUSHEK-BOURY, NANCY - IA STATE UNIV., AMES, IA
item Bosworth, Brad
item Stabel, Thomas
item Kehrli Jr, Marcus
item TAYLOR, MARGE - IA STATE UNIV., AMES, IA

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/6/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Circulating levels of endotoxin often rise during gram-negative bacteremia and after antibiotic therapy of sepsis. This rise is often followed by an increase in circulating TNF levels which can result in cachexia or death. We are interested in reducing the impact of bacterial infection due to excess TNF production in swine. Recombinant soluble TNF receptors (sTNFR) protect against lethal effects of endotoxemia by binding TNF in circulation, thus preventing its association with TNF receptors on target cells. Since TNF receptors bind to TNF in a species specific fashion, a clone encoding the entire extracellular and transmembrane domains was isolated from a porcine lung cDNA library. We subcloned the region encoding the mature extracellular domain into a pFLAG expression vector and transformed it into Escherichia coli strain DH5alpha. Expressed protein was a FLAG-pTNFR1 fusion, under control of an inducible promoter. Presence of the FLAG peptide allowed purification of protein using an anti-FLAG immunoaffinity column, resulting in an average yield of 120-150 ug of fusion protein per liter of culture. Human and murine sTNFR at high concentrations have been reported to inhibit bioactivity of TNF, and at lower concentrations to enhance TNF bioactivity.