Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus

摘要

The spike (S) glycoprotein is one of the major structure proteins of SARS-associated coronavirus (CoV). Fragment 450-650 (S450-650) of the S protein contains receptor-binding domain and neutralizing epitopes. In this study. S450-650 was expresssed with a histidine tag in Escherichia coli BL21. Bacterial inclusion bodies containing the recombinant S450-650 were solubilized with 8 M urea and then applied onto a Ni-nitrilotriacetic acid column. On-column refolding and purification was performed. Reduced glutathione, and oxidized glutathione were included in the refolding buffer. In the wash and elution buffers. glycerol and glucose were necessary. T additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 mug/ml in soluble form, which maintained appropriate antigenicity and immunogenicity. It was able to induce strong IgG responses in BALB/c mice. In Western blot assays, the recombinant S450-650 was recognized by monoclonal Ab against the His-tag and also sera from a convalescent SARS patient. S450-650-based ELISA system was able to detect anti-SARS-CoV IgG Abs in patient sera. (C) 2004 Elsevier Inc. All rights reserved.