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.
Article
in Zh
| WPRIM
| ID: wpr-249593
Responsible library:
WPRO
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.
Full text:
1
Database:
WPRIM
Main subject:
Recombinant Fusion Proteins
/
Immunoglobulin Variable Region
/
Immunoglobulin Fc Fragments
/
Antigens, Differentiation
/
Antigens, CD
/
Cloning, Molecular
/
Allergy and Immunology
/
Escherichia coli
/
CTLA-4 Antigen
/
Genetics
Limits:
Humans
Language:
Zh
Journal:
Journal of Biomedical Engineering
Year:
2006
Document type:
Article