HIGHLY EFFICIENT RECOVERY OF FUNCTIONAL SINGLE-CHAIN FV FRAGMENTS FROM INCLUSION-BODIES OVEREXPRESSED IN ESCHERICHIA-COLI BY CONTROLLED INTRODUCTION OF OXIDIZING REAGENT - APPLICATION TO A HUMAN SINGLE-CHAIN FV FRAGMENT

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coil w as developed. Stepwise removal of denaturing reagent and controlled ad dition of oxidizing reagent were found to be the most effective condit ions to achieve for almost complete recovery of functional monomeric s cFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chai n Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHE L10, and a human monoclonal antibody against the D antigen of the Rh b lood group, D10, in solubilized inclusion bodies could be refolded und er these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments. (C) 1998 Elsevier Science B.V. All rights reserved.