[The study of in vitro refolding conditions for humanized anti-CTLA4-scFv expressed in E. coli].
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.
Zeng
Lingyu
L
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital of Sichuan University, Chengdu 610041, China.
Huang
Qiang
Q
Chen
Lihong
L
Wan
Lin
L
Lu
Xiaofeng
X
D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D060908:CTLA-4 Antigen / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D057127:Single-Chain Antibodies / Q000737:chemistry*
D061067:Antibodies, Monoclonal, Humanized; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D057127:Single-Chain Antibodies