Journal Article

Refolding and Recovery of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) from Inclusion Bodies Expressed by <i>Escherichia coli</i>

Hiroshi Oneda and Kunlyo Inouye

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 126, issue 5, pages 905-911
Published in print November 1999 | ISSN: 0021-924X
Published online November 1999 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022533
Refolding and Recovery of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) from Inclusion Bodies Expressed by Escherichia coli

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The recombinant prepro-fonn 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*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*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 (kcat/Km) of matrilysin was1.7X105 M-1.s-1.

Keywords: inclusion bodies; martrilysin; matrix metalloprpteninase; proteolysis; refolding

Journal Article.  0 words. 

Subjects: Biochemistry

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