Construction of a VEGFR1 and VEGFR2 Bi-targeting oligopeptide-based fusion protein A7/G6-LDP and its anti-angiogenic activity / 中国药学杂志

ABSTRACT

OBJECTIVE: To construct a VEGFR1 and VEGFR2 bi-targeting oligopeptide-based fusion protein A7/G6-LDP and investigate its anti-angiogenic activity and the mechanism of action. METHODS: PCR and overlap PCR were used to construct the fusion protein A7/G6-LDP expression vector that consists of the gene encoding G6, A7, LDP, and the linker peptide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC. The binding activity to endothelial cells was examined by ELISA and immunocytochemical staining. The inhibition of HMEC-1 proliferation was determined with CCK-8 assays. The phosphorylation of AKT and c-Raf was detected by Western blotting. HMEC-1 migration was determined using a wound healing assay and tube formation was measured after incubation on Matrigel. RESULTS: The data of DNA sequence confirmed that the A7/G6-LDP fusion protein was correctly constructed. The fusion protein was recovered in high yield (up to 20 mg·L-1) and high purity after His-tag purification. A7/G6-LDP bound to HMEC-1 and HUVEC, respectively; in addition, it inhibited endothelial cell proliferation more effectively than LDP alone when used higher concentration. Moreover, A7/G6-LDP disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. The mechanistic study showed that A7/G6-LDP decreased the phosphorylation of AKT in HMEC. CONCLUSION: The engineered VEGFR1 and VEGFR2 bi-targeting oligopeptide-based fusion protein A7/G6-LDP effectively inhibits anti-angiogenesis. It might serve as a drug delivery carrier in targeted cancer therapy.