Purification of an anti-HBsAg scFv and measurement of its affinity constant

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, Ni²⁺ 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) ×10⁷ 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.