Journal Article

A33scFv–Green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

Ulf Petrausch, Jens Dernedde, Vânia Coelho, Hossein Panjideh, Dietmar Frey, Hendrik Fuchs, Eckhard Thiel and P. Markus Deckert

in Protein Engineering, Design and Selection

Volume 20, issue 12, pages 583-590
Published in print December 2007 | ISSN: 1741-0126
Published online November 2007 | e-ISSN: 1741-0134 | DOI:
A33scFv–Green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

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Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv–green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by ∼95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.

Keywords: ADEPT; A33 antibody; colon carcinoma; recombinant fusion proteins; tumor targeting

Journal Article.  4874 words.  Illustrated.

Subjects: Proteins

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