Production of fluorescent single-chain antibody fragments in Escherichia coli

We describe a novel vector-host system suitable for the efficient preparati on of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expre ssion of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein ( GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by an alyzing of the typical GFP fluorescence of the transformed cells under UV i llumination. The brightest signal was obtained when scFv1F4 was linked to t he cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 exp ressed under these conditions did not contain disulfide bridges, about 1% o f the molecules were able to bind antigen. Fluorescence analysis of antigen -coated agarose beads incubated with the cytoplasmic scFv-GFP complexes sho wed that a similar proportion of fusions retained both E6-binding and green -light-emitting activities. The scFv1F4-GFPuv molecules were purified by af finity chromatography and successfully used to detect viral E6 protein in t ransfected COS cells by fluorescence microscopy. When an anti-beta-galactos idase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amou nts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays. (C) 2000 Academic Press.