Matrix-assisted refolding of single-chain Fv-cellulose binding domain fusion proteins

We describe a method for the isolation of recombinant single-chain antibodi es in a biologically active form, The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as in soluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic sol vent, then reversibly immobilized onto a cellulose matrix via specific inte raction of the matrix with the cellulose binding domain (CBD) moiety. The e fficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustn ess of the Clostridium thermocellum CBD we use. This CBD is unique in retai ning its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of th e fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolde d single-chain antibodies in their native state are then recovered by relea sing them from the cellulose matrix in high yield of 60% or better, which i s threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have gener al applicability for the production of many protein-CBD fusions in which th e fusion partner is insoluble upon expression. (C) 1999 Academic Press.