Expression Of Recombinant Human Soluble Platelet-Derived Growth Factor Receptor Beta/Fc Chimera In Insect Cell

Platelet-derived growth factors (PDGFs), which has the ability to induce lymphangiogenesis and to promote lymphatic metastasis, have shown as a potent drug target to against tumor. Soluble platelet-derived growth factor receptor (sPDGFR), which exerts biological effects by blocking the function of PDGF through competing with membrane PDGFR (mPDGFR) to bind PDGF, can be promising specific PDGF antagonist. However, the native sPDGFR has short half life and low affinity in binding PDGF. In the present study, we expressed a fusion protein sPDGFRβ/Fc, combining the extracellular domain of PDGFR β chain and the Fc region of human IgG.Using Bac-to-Bac system, we generated a recombinant baculovirus. Firstly, the gene PDGF Rβ/Fc was cloned into a pFastBac vector. Secondly, the recombinant plasmid pFastbac-sPDGFRβ/Fc was transformed into DH10Bac competent cells. Through site-specific transposition, the rBacmid-sPDGFRβ/Fc was obtained. Finally, the complete virus was generated by transfecting the rBacmid-sPDGFRβ/Fc DNA into insect cell Sf9.The Sf9 cells which infected with complete virus were adapted to serum-free medium SF-900 II, and the sPDGFRβ/Fc can be released into medium. Through purification by Protein A affinity chromatography, 75% pure sPDGFRβ/Fc was obtained with a yield of 1 mg/L and was confirmed that can inhibit PDGF-induced proliferation. We also developed the suspension culture system of Sf9 in serum free medium, showing that the supplementation of glucose to both adherent culture cell and suspension culture cell resulted in the promotion of cell growth and higher cell yield.In conclusion, this study has set the stage for a continued development of protein sPDGFRβ/Fc for future research.