[Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli].
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.
Huang
Qiang
Q
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, China.
Chen
Lihong
L
Zeng
Lingyu
L
Wan
Lin
L
Li
Shengfu
S
Lu
Xiaofeng
X
Cheng
Jingqiu
J
D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D060908:CTLA-4 Antigen; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins