The study of in vitro refolding conditions for humanized anti-CTLA4-scFv expressed in E. coli.
- Author:
Lingyu ZENG
1
;
Qiang HUANG
;
Lihong CHEN
;
Lin WAN
;
Xiaofeng LU
Author Information
1. Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital of Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Antibodies, Monoclonal, Humanized;
chemistry;
CTLA-4 Antigen;
chemistry;
Escherichia coli;
metabolism;
Humans;
Inclusion Bodies;
chemistry;
Protein Folding;
Single-Chain Antibodies;
chemistry
- From:
Journal of Biomedical Engineering
2005;22(3):588-592
- CountryChina
- Language:Chinese
-
Abstract:
The purpose of the study is to explore the in vitro refolding protocol for humanized anti-CTLA4-scFv expressed in E. coli. The inclusion bodies are denatured and then diluted or dialyzed into a refolding buffer. We analyzed several factors affecting the refolding yield, including refolding time, temperature, and redox environment. The refolded target proteins are analyzed by non-reducing SDS-PAGE, and the concentration of refolded proteins are examined by Bio-Rad Dc Protein Assay kit. The result shows that a high yield of the protein with natural conformation can be acquired in the condition of 0.15 mol x L(-1) sodium chloride, 50 mmol x L(-1) Tirs-HCl, pH 8.0 buffer containing 1 micromol x L(-1) reduced glutathione and 3 micromol x L(-1) oxidized glutathione. The refolding time is 48 to 54 h at 4 degrees C. 28 mg refolded proteins are produced from 3.9g E. coli.
