PEDS Advance Access originally published online on November 22, 2007
Protein Engineering Design and Selection 2007 20(12):583-590; doi:10.1093/protein/gzm043
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A33scFv–Green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting
1Medizinische Klinik III—Hematology, Oncology und Transfusion Medicine 2 Institute for Clinical Chemistry and Pathobiochemistry, Charité—Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany
3 To whom correspondence should be addressed. E-mail: markus.deckert{at}charite.de
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
Received February 28, 2007; revised July 3, 2007; accepted July 6, 2007.