Fluobodies: green fluorescent single-chain Fv fusion proteins

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent prot eins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selec ted recombinant antibodies by flow cytometry or fluorescent cell Staining. Two different single-chain variable fragment antibodies, both directed agai nst the lipopolysaccharide of the bacterium Ralstonia solanacearum have bee n genetically fused to a red-shifted green fluorescent protein and the prod uced fusion protein rested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affini ty chromatography. They function well in flow cytometry and immunofluoresce nt cell staining, are specific for their target antigens and, unlike FITC-c onjugated antibodies, they do not fade upon illumination. (C) 1999 Elsevier Science B.V. All rights reserved.