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Protein Engineering, Vol. 13, No. 6, 445-452, June 2000
© 2000 Oxford University Press

Green fluorescent antibodies: novel in vitro tools

Joanne L. Casey, Andrew M. Coley, Leann M. Tilley and Michael Foley1

Department of Biochemistry and CRC for Diagnostic Technologies, La Trobe University, Bundoora, Victoria, 3083, Australia

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.coli. In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.coli cells. Further characterization revealed that the periplasmic N'-terminal EGFP–scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP–scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP–scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.


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