Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus.
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 expressed 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 additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 microg/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.
Zhao
Jin-Cun
JC
Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. zhaojincun@hotmail.com
Zhao
Zhen-Dong
ZD
Wang
Wei
W
Gao
Xiao-Ming
XM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005260:Female; D005620:Freund's Adjuvant / Q000494:pharmacology; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007114:Immunization; D007117:Immunization, Secondary; D007279:Injections, Subcutaneous; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D014170:Transformation, Genetic
D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006639:Histidine; D005620:Freund's Adjuvant