title: Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1.
abstract: Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19 × 10(-8) M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35 × 10(-7) M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
authors.affiliation: Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.affiliation: Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
authors.forename: G M
authors.initials: GM
authors.lastname: Wu
authors.affiliation: Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
authors.forename: Y H
authors.initials: YH
authors.lastname: Yue
authors.affiliation: Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
authors.forename: G L
authors.initials: GL
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000936:Antigen-Antibody Complex; D002448:Cell Adhesion; D002845:Chromatography; D003956:Dialysis; D054644:Ear Auricle / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000494:pharmacology; D002479:Inclusion Bodies / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000187:drug effects / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D051379:Mice; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods; D058849:Protein Refolding; D020673:Protein Renaturation; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000494:pharmacology; D014992:Xylenes / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007128:Immunoglobulin Fragments; D057127:Single-Chain Antibodies; D014992:Xylenes; D018799:Intercellular Adhesion Molecule-1