Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I》.pdf

Journal of Biotechnology 77 (2000) 169–178 locatejbiotec Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I Won-Kyung Cho a, Uik Sohn b, Ju-Won Kwak a,* a Cardioascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Taejon 305-600, Republic of Korea b Genetic Engineering Department, Kyungpook National Uniersity, Taegu 701 -702, Republic of Korea Received 30 March 1999; received in revised form 23 September 1999; accepted 4 October 1999 Abstract An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (1, ), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (V ) and light chain (V ) were connected by a (Gly Ser) H L 4 3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatogra- phy. The purity and activity of purified scFv were con