The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13).

We have cloned, expressed, and purified a recombinant C-terminal truncated form (residues Glu103-Asn274) of human collagenase-3 (MMP-13) in Escherichia coli. The molecule contains the catalytic domain of the enzyme and is expressed almost exclusively as inclusion bodies. Using a combination of rapid dilution and diafiltration, the enzyme has been successfully refolded from these inclusion bodies. The protein was purified to homogeneity using cation-exchange and size-exclusion chromatography. The purified enzyme is a monomer with a Mr of approximately 19,600 and was characterized using a variety of techniques including, SDS-PAGE, RP-HPLC, LC-MS, amino acid analysis, and dynamic light scattering. Microheterogeneity at the NH2-terminus of the refolded, purified protein disappeared after incubating for 30-60 min at 37 degreesC. The enzyme was highly active using a fluorescent peptide substrate and was found to release S-GAG from bovine nasal cartilage chips.