Refolding and recovery of recombinant human matrix metalloproteinase 7 (matrilysin) from inclusion bodies expressed by Escherichia coli.
The recombinant prepro-form of human matrix metalloproteinase 7 (matrilysin or MMP-7) was overexpressed in Escherichia coli as insoluble inclusion bodies. The recombinant protein was refolded by 100-fold dilution after solubilization with 6 M guanidine HCl. The refolding was monitored by the recovery of matrilysin activity. The addition of either 1.0 M arginine or 0.1% Brij-35 promoted remarkably the refolding. The refolding was dependent on pH and temperature, with lower temperature (<10 degrees C) and pH 6-8 preferable. Glutathione had no effect on refolding, and it was excluded from the refolding conditions. Starting with inclusion bodies (2.0 g, wet) containing 360 mg protein, 29.5 mg of pro-matrilysin (30 kDa) was obtained after refolding with 1.0% Brij-35 at pH 7.5 and 4 degrees C for 12 h. Pro-matrilysin (24.0 mg) was purified to homogeneity by cation-exchange HPLC with a 15-fold increase in purity and an activity yield of 81.3%. Pro-matrilysin was converted entirely to matrilysin (19.0 kDa; 15.2 mg) by activation with a mercuric reagent. The activity (k(cat)/K(m)) of matrilysin was 1.7 x 10(5) M(-1) x s(-1).
Oneda
H
H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Inouye
K
K
D001120:Arginine; D003902:Detergents; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000077423:Polidocanol; D011092:Polyethylene Glycols; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D003902:Detergents; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D000077423:Polidocanol; D011092:Polyethylene Glycols; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D001120:Arginine; D008666:Metalloendopeptidases; C099583:promatrilysin; D020783:Matrix Metalloproteinase 7