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-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length huma...

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Published inBiochemical and biophysical research communications Vol. 216; no. 1; pp. 329 - 337
Main Authors Ohare, M.C., Curry, V.A., Mitchell, R.E., Cawston, T.E.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 02.11.1995
Subjects
Alanine
Amino Acid Sequence
Base Sequence
Cloning, Molecular
Collagenases - biosynthesis
Collagenases - chemistry
Collagenases - metabolism
DNA Primers
DNA, Complementary
Electrophoresis, Polyacrylamide Gel
Enzyme Stability
Escherichia coli
Glutathione Transferase - biosynthesis
Humans
Isoleucine
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Point Mutation
Polymerase Chain Reaction
Proline
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - isolation & purification
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
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Summary:During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr 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.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1995.2628