Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli.
- Author:
Qiang HUANG
1
;
Lihong CHEN
;
Lingyu ZENG
;
Lin WAN
;
Shengfu LI
;
Xiaofeng LU
;
Jingqiu CHENG
Author Information
1. Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Antigens, CD;
biosynthesis;
genetics;
immunology;
Antigens, Differentiation;
biosynthesis;
genetics;
immunology;
CTLA-4 Antigen;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Immunoglobulin Fc Fragments;
biosynthesis;
genetics;
immunology;
Immunoglobulin Variable Region;
biosynthesis;
genetics;
immunology;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology
- From:
Journal of Biomedical Engineering
2006;23(2):388-391
- CountryChina
- Language:Chinese
-
Abstract:
To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.
