Abstract
Recombinant human transforming growth factor alpha (TGF alpha), which is active as assessed by competition with epidermal growth factor (EGF) for binding to the EGF receptor, has been produced in Escherichia coli and separated from misfolded and inactive forms of recombinant TGF alpha using reverse-phase high performance liquid chromatography. The purified recombinant TGF alpha was used to produce a monoclonal antibody that binds to active TGF alpha specifically. The antibody was coupled to Sepharose and used as an independent method for purifying active TGF alpha. The EGF receptor binding activity of antibody affinity purified TGF alpha is comparable to that of high performance liquid chromatography-purified active TGF alpha, and is 0.55 mg of EGF eq/mg of TGF alpha. The disulfide arrangement of the active TGF alpha was determined after digestion with thermolysin, and found to be analogous to the disulfide arrangement previously determined for EGF (Savage, C. R., Hash, J. H., and Cohen, S. (1973) J. Biol. Chem. 248, 7666-7672).
Highlights
Recombinant human transforming growth factor a (TGFa),which is active as assessed by competitionwith epidermal growth factor (EGF) for binding to the EGF receptor, has been produced in Escherichia coli and separated from misfolded and inactive forms of recombinant Transforminggrowthfactor a (TGFa) using reverse-phase high performance liquid chromatography
We have determined the specific activity of recombinant human TGFapurified by HPLC and compared it to the specific activity of recombinant TGFa purified by immunoaffinity chromatography.The specific activities of these preparationsagree within experimental error, suggesting that each of these procedures eliminates inactive
TGFa has bseheonwn to compete with epidermal growth factor (EGF) for binding to the EGF receptor(1,7, 8)
Summary
Purification of Recombinant TGFa.-TGFa was expressed as a trp LE fusion protein (fusTGFa) in E. coli containing the plasmid pTE5 (9). 30 g of E. coli paste were suspended in 200 ml of lysis buffer Purification of Recombinant TGFa.-TGFa was expressed as a trp LE fusion protein (fusTGFa) in E. coli containing the plasmid pTE5 (9). Pellet containing the fusTGFa in refractile bodies was collected by centrifugation at 4700 X g for 15 min and was resuspended in 40 ml of lysis buffer in which lysozymehad been omitted. The cDNA codingfor TGFa was expressed inEscherichia coli pellet was layered over 160 mlof a cold 40% glycerol solution and as a fusion protein, in which a 17-residue portion of the E . Fractions were pooled based on their specific activity as determined by amino acid composition and the.
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