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Construction of Fc fusion protein EVP1 targeting members of VEGF-EGFR and its binding affinity
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Abstract:
Objective: To construct the Fc fusion protein EVP1 with high affinity to bind epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and human epidermal growth factor receptor 2 (HER2), and to analyze its multi-target binding affinity for further study of antitumor therapy. Methods: The coding sequences of Herin and FLT-1D2 (the second Ig-like domain of FLT-1gene) were amplified by PCR individually, and joined to synthesize human IgG1 Fc fragment with point mutations via overlap PCR. Then, the fused open reading frame of Herin-Flt-1D2-Fc (named as EVP1) was inserted into pDC659 plasmid, an adenoviral shuttle vector. The plasmid of pDC659-EVP1 and pPE3-F35 were co-transfected into 293 cells to package Ad5/35-EVP1, a replication-incompetent adenovirus carrying the EVP1 coding gene. After identification, amplification, purification and titration determination, the recombinant virus Ad5/35-EVP1 was used to transfect 293 cells at a MOI=11. Four days post transfection, culture supernatant was collected to enrich and purify the fusion protein EVP1 through ammonium sulfate salting out method and protein G affinity chromatography. Subsequently, Western blotting and ELISA were performed to detect the expression of EVP1. Finally, the binding affinities of EVP1 to EGFR, HER2 and VEGF were both qualitatively identified via indirect immunofluorescent assay and quantitatively determined on the Octet Red 96 Biomolecular Interaction Analysis System. Results: The recombinant adenovirus Ad5/35-EVP1 was constructed successfully, and its titer was 5.4×109 PFU/ml. With the expression system of Ad5/35-EVP1 and 293 cells, the fusion protein EVP1 could be effectively produced with the expression level being (1 613.94?24.65) ng/ml when MOI=11. The obtained EVP1 could bind EGFR on A431 cells and HER2 on SK-OV-3 cells with high affinity, and the affinity constants Kd of EGFR, HER2 and VEGF were 4.55,18.70, and 0.63 nmol/L, respectively. Conclusion: The fusion protein EVP1 was constructed successfully, and EVP1 has high binding-affinity to VEGF and EGFR family members, showing a good clinical potent in targeted cancer therapy.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81001013), and the Excellent Academic Leaders of Shanghai (No. 10XD1406500)
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