Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli

• Recombinant anti-rabies scFv57RN was expressed in bacteria in inclusion bodies.
• Ion-exchange column chromatography yielded scFv57RN inclusion bodies with 95% purity.
• On-column refolding showed advantages over dialysis and dilution methods.
• scFv57RN showed high neutralization in vitro and protected mice from rabies virus.

An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.