Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Brazilian Journal of Medical and Biological Research

H. Sun¹  G.m. Wu¹  Y.y. Chen¹  Y. Tian¹  Y.h. Yue¹  G.l. Zhang¹ 

关键词: Intercellular adhesion molecule-1;    Single-chain variable antibody fragment;    Expression;    Purification;    Renaturation;    Biological activity;   

DOI  :  10.1590/1414-431X20143276

来源: SciELO

【 摘 要 】

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.

【 授权许可】

CC BY   
 All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License