Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58.
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 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.
Chadwick
M P
MP
Department of Pathology, University of Newcastle upon Tyne, Royal Victoria Infirmary, U.K.
May
F E
FE
Westley
B R
BR
D000595:Amino Acid Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012694:Serine / Q000235:genetics; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins
D009363:Neoplasm Proteins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins; D012694:Serine; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol