PMID: 8948462Jun 15, 1995Paper

Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

The Biochemical Journal
M P ChadwickB R Westley

Abstract

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same appa...Continue Reading

Citations

Nov 26, 1997·The Journal of Pathology·F E May, B R Westley
Aug 26, 1998·The Journal of Pathology·T MarchbankR J Playford
Oct 24, 2006·Annals of the New York Academy of Sciences·Lothar Steidler, Pieter Rottiers
Jan 1, 1997·Journal of Clinical Gastroenterology·I M Modlin, R Poulsom
Nov 22, 2005·Expert Opinion on Drug Delivery·Lothar Steidler
Jul 31, 1998·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·N A Wright
Mar 29, 2002·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Sara J PrestBruce R Westley
Mar 28, 1997·Journal of Molecular Biology·V I PolshakovJ Feeney
Sep 18, 1998·Biochimica Et Biophysica Acta·S RibierasM C Rio
Jan 1, 1995·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·F E May, B R Westley
Mar 1, 1996·Baillière's Clinical Gastroenterology·R Poulsom

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