PURIFICATION OF HUMAN MATRILYSIN PRODUCED IN ESCHERICHIA-COLI AND CHARACTERIZATION USING A NEW OPTIMIZED FLUOROGENIC PEPTIDE SUBSTRATE

Human promatrilysin (matrix metalloproteinase-7 has been produced in E scherichia coli as an N-terminal fusion protein with ubiquitin. The in soluble product was solubilized, refolded, and activated with amino-ph enylmercuric acetate. Activation of the fusion protein demonstrated ki netics and intermediates that were very similar to those observed duri ng activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to >95% homogene ity using a Sepharose-Pro-Leu-Gly-NHOH affinity column, The matrilysin purified by this procedure is indistinguishable from the enzyme purif ied from CHO cells with respect to the kinetic parameters for hydrolys is of a peptide substrate and the ability to obtain diffraction qualit y crystals in the presence of an inhibitor of the enzyme. Additionally , to facilitate detailed kinetic analyses of matrilysin, a new fluorog enic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala -Leu-Trp-Arp-Ser (Dnp, dinitrophenyl) has been synthesized. This pepti de is the best substrate developed for matrilysin thus far with K-m an d k(cat) values of 26 mu M and 5.0 s(-1), respectively. (C) 1995 Acade mic Press, Inc.