Purification of Human Matrilysin Produced inEscherichia coliand Characterization Using a New Optimized Fluorogenic Peptide Substrate

• Published in: Archives of biochemistry and biophysics 1995-12, Vol.324 (1), p.59-64
• Main Authors: Welch, Anthony R., Holman, Christopher M., Browner, Michelle F., Gehring, Michael R., Kan, Chen-Chen, Van Wart, Harold E.
• Format: Article
• Language: English
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1995.9929

Human promatrilysin (matrix metalloproteinase-7) has been produced inEscherichia colias an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with aminophenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to >95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far withKmandkcatvalues of 26 μMand 5.0 s−1, respectively.