Mc. Ohare et al., STABILIZATION OF PURIFIED HUMAN COLLAGENASE BY SITE-DIRECTED MUTAGENESIS, Biochemical and biophysical research communications, 216(1), 1995, pp. 329-337
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 c
leavage site has been identified (Pro269- Ile270) and in an attempt to
obtain full-length human collagenase (i.e.,Mr 42570), this cleavage s
ite and another potential cleavage site (Ala258- Ile259) have been mut
ated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutate
d cDNA was then cloned into the expression vector, pGEX2T, and express
ed in Escherichia coli as a fusion protein with glutathione-S-transfer
ase (GST). After cleavage with factor Xa, the mutated collagenase was
purified on a peptide hydroxamic acid affinity column. The mutated rec
ombinant collagenase is stable, remains full length and retains the ab
ility to cleave collagen. (C) 1995 Academic Press, Inc.