Fluobodies: green fluorescent single-chain Fv fusion proteins

✍ Scribed by Remko A Griep; Charlotte van Twisk; Jan M van der Wolf; Arjen Schots

• Book ID: 114052255
• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 469 KB
• Volume: 230
• Category: Article
• ISSN: 0022-1759
• DOI: 10.1016/s0022-1759(99)00131-3

✦ Synopsis

An expression system pSKGFP , which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.