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 Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric 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 with Km and kcat values of 26 microM and 5.0 s-1, respectively.
Welch
A R
AR
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto.
Holman
C M
CM
Browner
M F
MF
Gehring
M R
MR
Kan
C C
CC
Van Wart
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003460:Crystallization; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
C097064:2,4-dinitrophenyl-arginyl-prolyl-leucyl-alanyl-leucyl-tryptophyl-arginyl-serine; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7