Purification of human single-chain variable fragment of anti-digoxin antibody expressed in escherichia coli by anion-exchange chromatography in a fast protein liquid chromatography system

Objective: To explore the purification conditions of human single-chain variable fragment of anti-digoxin antibody （ADAscFv） expressed in escherichia coli（E.Coli） by anion-exchange chromatography. Methods: ADAscFv was expressed into the culture medium as soluble protein in E.coli strain HB2151; ADAscFv in the culture medium supernatant was salted out with 50% ammonium sulfate; ADAscFv was eluted by Tris-HCl buffers containing NaCl continuous concentration gradient and NaCl step-wise concentration gradient from anion-exchange chromatographic column in a fast protein liquid chromatography（FPLC） system;the antigen-binding activity of ADAscFv of the recovered samples was analyzed by ELISA,and the purity was analyzed by SDS-PAGE. Results: The elution profile demonstrated that there were six protein peaks;Of all the samples, the antigen-binding activity of ADAscFv of the sample recovered from the second peak was the highest as showed by ELISA;the electrophoretogram of the sample recovered from the second peak showed a typical pattern of one band representing ADAscFv. Conclusions: ADAscFv expressed into the culture medium as soluble protein in E.coli could be efficiently purified by anion-exchange chromatography in the FPLC system,the isolated ADAscFv has a high purity,therefore,it may be used to antagonize the digoxin toxication or overdose.