Optimized refolding and characterization of active C-terminal ADAMTS-18 fragment from inclusion bodies of Escherichia coli

Abstract

The human ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin type-1 modules 18) is a new member of the ADAMTS family. The C-terminal ADAMTS-18 fragment is highly effective at promoting platelet thrombus dissolution in murine model of ischemic stroke, showing significant clinical relevance. In this report, the C-terminal ADAMTS-18 fragment with a GST tag (named rADAMTS-351) was overexpressed mainly as inclusion bodies in Escherichia coli BL21 (DE3) pLysS. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The optimal buffers for refolding rADAMTS-351 was composed of 50mM Tris–HCl buffer at pH 8.0, 5mM EDTA, 150mM NaCl, 0.1mM DTT, 1mM GSH, and 0.2mM GSSG. The refolded rADAMTS-351 was dialyzed and further purified by glutathione-agarose beads. The purity of the final product reached 98% as evaluated by SDS–PAGE and Coomassie Brilliant Blue R250 staining. The recombinant protein displayed its immunoreactivity with anti-C-terminal ADAMTS-18 antibodies by Western blotting. Mass spectroscopic analysis indicated a molecular mass of 65,327Da as theoretically expected. Purified rADAMTS-351 displayed its bioactivity by inducing platelet fragmentation, which ranged from 81–96% compared to active C-terminal ADAMTS-18 standards. The expression and refolding strategy described in this study allows convenient small-scale production of rADAMTS-351 with biological function and therapeutic potential.