Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Abstract

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18°C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase.

Fig. 1

Expression vector map showing the major restriction enzyme cleavage sites, transcription control region, and antibiotic resistance information. After subcloning the resulting plasmid had 7.205 bps.

Fig. 2

Expression of rhMMP-2 in BL21(DE3)/pLysS. Cytoplasmic supernatants were collected after different times of induction by IPTG. The rhMMP-2 activity was detected in the soluble fraction in neutral pH buffer after sonication of the bacterium pellet. Gelatinolytic activity was detected by a fluorimetric assay and is expressed as mean and S.D. (A) and in a gelatin zymogram (B). Recombinant protein expression was induced with 100 μM IPTG, and bacteria were harvested after different induction times, as indicated. Bacteria were grown at 18 °C and 180 rpm. RFUs: relative fluorescence units.

Fig. 3

Gelatin affinity purification of rhMMP-2 produced from BL21(DE3)/pLysS. The rhMMP-2 was purified on a gelatin-sepharose column as described in Section 2, samples from each step were analyzed in a 12% SDS-PAGE gel, and proteins were stained with Coomassie Blue. Lane 1, molecular weight markers (in kDa); lane 2, BL21(DE3)/pLysS whole pellet; lane 3, soluble fraction after cells were sonicated; lane 4, unbound fraction; lane 5, wash fraction; lane 6, relatively pure rhMMP-2 (72 kDa) eluted with 5% DMSO. Western blotting (Wb) confirmed the identity of the rhMMP-2 and revealed the presence of some possible degradation products of the full-length protein.