Biopolymers and cell. 2008; 24 (1): 51 - 59

Refolding of ScFv-CBD fusion protein from Escherichia coli inclusion bodies

O. B. Gorbatuk, U. S. Nikolayev, D. M. Irodov, I.Ya. Dubey, P. V.Gilchuk

An inexpensive and effective refolding method for ScFv-CBD fusion protein recovery from E. coli inclusion bodies has been developed. The principle of the proposed method is the stepwise dilution of solubilized ScFv-CBD in the presence of 6 M guanidine hydrochloride with refolding buffer containing L-arginine and glutathione. The influence of initial protein concentration and the molar ratio of oxidized and reduced form of glutathione that was introduced at certain refolding stages has been carefully investigated. It was shown that the decrease in the yield of functional ScFv-CBD and formation of significant amount of soluble aggregates take place when the initial protein concentration is higher then 0.53 mg/ml. The optimal refolding conditions providing highest yield of soluble monomeric ScFv-CBD as well as the highest functional activity of antigen-binding (ScFv) and cellulose-binding (CBD) moieties (~63 % and ~96 %, respectively) have been found.

Keywords: fusion protein, inclusion bodies, refolding, single-chain antibodies, cellulose-binding domain.