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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.
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