The Studies On High-level Expression Of Single Chain Antibody Fusion Protein, ScFv-9R In Escherichia Coli And It Delivers SiRNA Into RT112Cells

Fibroblast growth factor receptor3(FGFR3) is a noted proto-oncogene involvedin the pathogenesis of many tumors, so more and more studies focus on the potentialuse of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In thisstudy, we designed a novel fusion protein containing the single-chain Fv (ScFv)against FGFR3and9-arginine, denoted as ScFv-9R. ScFv (single chain antibodyfragment) connected with VH (heavy chain variable region of an antibody) and VL(light chain variable region of an antibody) by a Linker (an about fifteen amino acidpeptide) still has characteristics of identifying antigen. The ScFv has the properties ofsmall size, high penetrability and low immunogenicity which enhance the potentialapplication value. Arginine is a basic amino acid and contains positive charge in theneutral environment which can combine with nucleic acid containing negative charge.Therefore, the nine-arginine peptide can combine with siRNA.The proteins expressed in traditional prokaryotic expression system can not foldand modify correctly, and exist in the precipitate in the form of inclusion bodieswhich are tough for the future study. To achieve the high-level production and solubleexpression, we established a soluble express system. ScFv and ScFv-9R were fusedwith small ubiquitin-related modifier (Sumo)by polymerase chain reactionandexpressed inEscherichia coliBL21(DE3). The recombinant bacteria was inducedby0.5mM isopropyl-β-D-thiogalactopyranoside for20h at20°C; Supernatants ofSumo-ScFvand Sumo-ScFv-9R were harvested and purified by DEAE Sepharose FFand Ni-NTA orderly, and supernatants of SumoScFv-9R was harvested and purifiedby Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9Rwas released from the fusion protein, respectively. The purity of ScFv or ScFv-9R wasshown to be higher than95%, and their yield reached about4mg per liter of bacterialculture. In vitro data showed that ScFv-9R can attenuate the phosphorylation ofFGFR3and ERKand inhabit the FGFR3path way in the absence or presence of FGF9.Gel retardation assay showed that1μg of ScFv-9R could efficiently bind to about4pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficientlybind and deliver siRNA into FGFR3-positive RT112cells but not intoFGFR3-negetive cells such as THP1cells.In conclusion, we use Sumo fusion system to acquire high-level production,soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel drug carrier, may have potential applications inantitumor studies and pharmaceutical development.