STABILIZATION OF PURIFIED HUMAN COLLAGENASE BY SITE-DIRECTED MUTAGENESIS

During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-M(r) fragment and a C-terminal 27000-M(r) fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e.,Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column. The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen. (C) 1995 Academic Press, Inc.