title: 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 50 mM Tris-HCl buffer at pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1 mM DTT, 1 mM GSH, and 0.2 mM 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 65,327 Da 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.
authors: Department of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China.
authors: Suying
authors: S
authors: Dang
authors: Tao
authors: T
authors: Hong
authors: Dawei
authors: D
authors: Bu
authors: Jian
authors: J
authors: Tang
authors: Jing
authors: J
authors: Fan
authors: Wei
authors: W
authors: Zhang
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000071096:ADAMTS Proteins; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010974:Platelet Aggregation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; C510099:ADAMTS18 protein, human