Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.
The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.
Kröger
M
M
Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany.
Tschesche
H
H
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D005780:Gelatin; D003094:Collagen; D017364:Collagenases; D020780:Matrix Metalloproteinase 9