High cytoplasmic expression in E-coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two indepe ndent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N -terminal encoding sequences related to human interleukin-2. Both fusion pr oteins were expressed insolubly and at high levels in the bacterial cytopla sm (approximately 30% of total bacterial protein in MM294 cells at a labora tory scale). When recombinant cells were cultured in 5-1 fermenters, expres sion and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. Afte r extraction and solubilization in urea, the cytoplasmic scFvs were purifie d using immobilized metal ion affinity chromatography, followed by DTT trea tment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of a n ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% far the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l(-1), respectively, for the af orementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant. (C) 1999 Published by Elsevier Science B.V. All r ights reserved.