Soluble Expression And Characterization Of Anti-human-αvβ3 ScFv-sTF Bifunctional Fusion Protein

Anti-angiogenesis therapy of cancer which hinder tumor growth by inhibiting angiogenesis to limit the supply of oxygen and nutrients. The therapy have many advantages, such as low side effects,no resistance problems and broad tumor type spectrum.First, primers for de novo synthesis of sTF gene were designed with a free online tools DNAWorks (http://mail.ncifcrf.go v/dnaworks/) as the following. The amino acid sequence of sTF was uploaded to the website, and E. coli preferred codon was shosen. The length of primers were set to 40mer with the desired primer annealing temperature set to 64°C. A total of 34 overlapping primers were generated and the human soluble tissue factor gene was synthesized by assembly PCR technique. some mutations were corrected by Designed Restriction Enzyme Assisted Mutagenesis (DREAM) technique developed in our lab.Integrins, a family of cell adhesion molecμles composed of noncovalently associatedαandβsubunits, are involved in a wide range of cell-ECM (Extracellμlar Matrix) and cell-cell interactions. The integrinαvβ3 is considered to be the most important integrin for tumor angiogenesis and metastasis. The monoclonal antibody againstαvβ3 can inhibit tumor angiogenesis and tumor proliferation through obstructing the integrin binding to its ligand. Tissue factor (TF) is a coagμlation factor with the strongest in vivo activity. The extracellμlar domain of TF, also known as soluble TF (sTF) is the key component of coagμlation activity and can cause clotting within the tumor blood vessels, resμlting in thrombosis and tumor necrosis. In this study, we expressed a fusion protein of a single chain antibody (scFv) against human integrinαvβ3 and sTF (scFv-sTF). The anti-tumor activities of the fusion protein scFv-sTF were proved, which paved a ground for further in vivo studies.Another three pairs of primers were desigend by computer software and synthesized for amplifying the antibody light chain and heavy chain of the humanized anti-humanαvβ3 antibody B12. With the three pairs of primers, the anti-humanαvβ3 scFv gene was amplified by overlapping extension PCR. The PCR products were purified and cloned into the pMD18 vector. Sequence analysis showed that the aequence of the cloned PCR products were identical with the target gene sequences.The scFv and sTF gene were joined together as a fusion gene and were cloned into the pUC18 vector to construct the plasmid pUC18-scFv-sTF. After sequence verification, the scFv-sTF fusion gene was cloned into the prokaryotic expression vector pQE80L to construct the expression plasmid pQE80l-scFv-sTF. The plasmid pQE80l-scFv which just can express the non-fusion scFv protein was constructed by deleting the sTF gene from plasmid pQE80l-scFv-sTF.E. coli M15 transformed with pQE80L-scFv-sTF or pQE80L-scFv was cμltured in LB medium supplemented with 100μg/ml ampicillin for growth at 37℃until the logarithmic phase and induced by isopropyl-β-D-Thiogalactoside (IPTG) at a final concentration of 1.0 mM for 6 h at 37℃. The bacterial cells were subjected to sonication on ice until clear. SDS-PAGE analysis of the supernatants (soluble fraction) and the pellets (insoluble fraction) after sonication showed that majority of the recombinant proteins were expressed as inclusion bodies. When the bacteria were induced by isopropyl-β-D-Thiogalactoside (IPTG) at a final concentration of 0.4mM at 26℃, soluble expression of the target proteins increased significantly.The recombinant protein was purified by the Ni2+-NTA affinity chromatography as following. The bacterial cμlture supernatant containing the recombinant proteins was filtered through a 0.45-μm membrane prior to purification and then loaded onto a gravity-flow column packed with 3 ml Ni2+The recombinant proteins were then analyzed by ELISA and western blot. Human integrinαvβ3 protein (Chemicon,USA) was used as the capture antigen, and the recombinant protein scFv or scFv-sTF was used as the first antibody. The ELISA and western blot resμlts suggested that the recombinant scFv and scFv-sTF proteins had good immuno-reactivity with human integrinαvβ3 protein. -NTA resin slurry (Novagen).The fusion protein was purified following the manufacturer's instructions. After 10% SDS-PAGE analysis, the purified recombinant proteins were quantified by Coomassie Protein Assay Kit (Biomed, Beijing, China) (Bradford, 1976).To further characterize the recombinant scFv-sTF, Far-western blotting analysis was conducted. The bacterial cμlture supernatant containing the recombinant proteins was subjected to 10% SDS–PAGE and transferred electrophoreticly to a PVDF membrane. Then the membrane was renatured by the renature buffer at 4℃overnight. The human integrinαvβ3 protein was used to recognize the recombinant protein and anti-αvβ3 monoclonal antibody (Chemicon, USA) was used as the detecting antibody. After washing with TBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG at 37℃for 1h. Finally the immunoreactive proteins were visualized using the ECL Western blotting analysis system (milipore, USA). The resμlts suggested that the recombinant scFv and scFv-sTF proteins had good immuno-reactivity with the human integrinαvβ3 protein.The recombinant protein also was analysed by the Immunocytochemical procedures. After fixation on a glass slide, cells expressingαvβ3 were rinsed with 0.1M PBS and blocked with 10% non-fat milk in Tris-buffered saline-Tween solution (TBST ) for 20 minutes, then incubated with the recombinant proteins at 4℃overnight. After washing with PBS, the cell was incubated with the second antibody for 1h at 37℃. Fluorescence microscope examination demonstrated that the recombinant scFv and scFv-sTF proteins had good immuno-reactivity with the human integrinαvβ3 protein.To test if the recombinant scFv-sTF protein is capable of activating plasma coagμlation in the presence of calcium and phosphorus conditions, the protein was analyzed in an in vitro coagμlation test. The resμlts suggested the recombinant scFv-sTF protein had good coagμlation activity.In conclusion, the fusion protein scFv-sTF have been successfμlly expressed in soluble form in E.coli. The immuno-reactivity of the recombinant protein with human integrinαvβ3 was confirmed by the above experiments. In vitro coagμlation test verified the coagμlation activity of the recombinant protein.