Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli / 生物医学工程学杂志
Journal of Biomedical Engineering
;
(6): 388-391, 2006.
Artigo
em Chinês
| WPRIM
| ID: wpr-249593
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Proteínas Recombinantes de Fusão
/
Região Variável de Imunoglobulina
/
Fragmentos Fc das Imunoglobulinas
/
Antígenos de Diferenciação
/
Antígenos CD
/
Clonagem Molecular
/
Alergia e Imunologia
/
Escherichia coli
/
Antígeno CTLA-4
/
Genética
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Biomedical Engineering
Ano de publicação:
2006
Tipo de documento:
Artigo
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