Gene expression, purification and characterization of recombinant human neutrophil collagenase

Abstract

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three α-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met 100 through Gly 262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca 2+ and Zn 2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20–25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the K i measurement against actinonin was similar to that of HNC prepared from human blood.