One‐step on‐column purification and refolding of a single‐chain variable fragment (scFv) antibody against tumour necrosis factor α

<jats:p>Single‐chain variable fragment (scFv) is a low‐molecular‐mass recombinant antibody and is usually expressed as inclusion bodies in <jats:italic>Escherichia coli</jats:italic>. Highly efficient purification and refolding methods are required to provide enough active proteins for therapeutic or diagnostic use. In the present study, an anti‐TNFα (tumour necrosis factor α) scFv (TNF‐scFv) was constructed and expressed in <jats:italic>E. coli</jats:italic> BL21(DE3) star as inclusion bodies, and a convenient procedure of one‐step on‐column purification and refolding was provided for it. Briefly, denatured TNF‐scFv was firstly captured by immobilized metal (Ni) affinity chromatography, and then non‐denaturing detergent (Triton X‐100)‐containing and β‐cyclodextrin‐containing solutions were loaded in turn on to the column to perform ‘artificial chaperone‐assisted refolding’ after removing impurities. More than 77% of denatured TNF‐scFv protein was refolded successfully with a purity of more than 95%. Activity assays showed that refolded TNF‐scFv could bind to rhTNFα (recombinant human TNFα) specifically with high affinity. It could inhibit rhTNFα from binding to TNF receptors and neutralize the cytolytic activity of rhTNFα against L929 cells effectively. A conclusion was obtained that this refolding method is time‐saving and suitable for industrial production. It may also be applicable to other scFvs or other recombinant proteins.</jats:p>