PRODUCTION AND COMPARISON OF MATURE SINGLE-DOMAIN TREFOIL PEPTIDES PNR-2/PS2 CYS(58) AND PNR-2/PS2 SER(58)

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, Cys(58), to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys(58) and pNR-2/pS2 Ser(58) proteins under non-denaturing conditions confirmed that the Ser(58) mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser(58) 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 Cys(58) treated with cysteine and untreated pNR-2/pS2 Ser(58) had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.