O'Hare M C, Curry V A, Mitchell R E, Cawston T E
Rheumatology Research Unit, Addenbrookes Hospital, Cambridge, United Kingdom.
Biochem Biophys Res Commun. 1995 Nov 2;216(1):329-37. doi: 10.1006/bbrc.1995.2628.
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.
在纯化过程中，人成纤维细胞胶原酶分解为两种主要形式，一种是N端22000/25000道尔顿的片段，另一种是C端27000道尔顿的片段；最可能的机制是自溶。裂解位点已被确定（Pro269-Ile270），为了获得全长人胶原酶（即42570道尔顿），该裂解位点和另一个潜在裂解位点（Ala258-Ile259）已通过PCR定向诱变进行突变：Ile270Ser和Ile259Leu。然后将突变的cDNA克隆到表达载体pGEX2T中，并在大肠杆菌中作为与谷胱甘肽-S-转移酶（GST）的融合蛋白表达。用因子Xa切割后，突变的胶原酶在肽羟肟酸亲和柱上纯化。突变的重组胶原酶是稳定的，保持全长并保留裂解胶原的能力。
