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

Abstract

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 approximately 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.

Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants

“…an OD 600nm of 2.0 to 2.5 at induction was optimal. This may be due to the susceptibility of our proteins to proteolytic cleavage at the linker site, as previous results have demonstrated [24]. As a method to limit proteolytic activity, lowering the production pH to around 3.0 has successfully been applied [26], but in our case, both proteins were highly sensitive to acidic conditions both in fermentation and purification.…”

“…The bifunctional activity of both fusion proteins from pilot expressions has recently been demonstrated in vitro [24,25], so in this paper we focus on optimization and upscaling of the fermentation method.…”

“…antibody binding and fluorescence) by direct flow cytometry and fluorescence microscopy as previously described [24]. For A33scFv::CDy, a previously described in-vitro application Bioprocess Biosyst Eng (2008) 31:559-568 561 of the complete ADEPT system in a fluorescein diacetate assay was employed [25].…”

“…To test for bifunctionality of the fusion protein, either direct immunofluorescence microscopy and flow cytometry [24] or a fluoresceine diacetate cytotoxicity assay were employed.…”

Produção de fragmento recombinante de anticorpo em Pichia pastoris

“…A maioria dos trabalhos utiliza o fenótipo Mut + (Li et al, 2007), embora alguns também utilizem o fenótipo Mut s (Wang et al, 1998;Shi et al, 2003;Damasceno et al, 2004;Gurkan, Symeonides e Ellar, 2004;Emberson et al, 2005;Petrausch et al, 2007;Pasman, Nagy e Kaushik, 2012).…”