Purification of an anti-HBsAg scFv and measurement of its affinity constant / 中国病理生理杂志
Chinese Journal of Pathophysiology
;
(12)1999.
Article
Dans Chinois
| WPRIM
| ID: wpr-519667
ABSTRACT
AIM:
To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6?His tag, and to determine the affinity constant of the purified recombinant product.METHODS:
Solubilizing in buffers containing urea or guanidine hydrochloride (GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni 2+ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA.RESULTS:
The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached (61.08?1 45)%. The affinity constant of the polished scFv was confirmed to be(2.30?0.32) ?10 7 L/mol.CONCLUSION:
The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro . The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
langue:
Chinois
Texte intégral:
Chinese Journal of Pathophysiology
Année:
1999
Type:
Article
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