Expression and purification of human vascular-endothelial-growth-factor-receptor-2 tyrosine kinase in Streptomyces for inhibitor screening

VEGF (vascular endothelial growth factor) is a critical regulator in angiogenesis through binding to its specific receptors, including VEGFR-2 (VEGF receptor 2), a kinase insert domain-containing receptor, on the surface of endothelial cells. As angiogenesis has been shown to be essential for malignancy of tumours, VEGFR-2 is a potential therapeutic target for the treatment of cancers. To explore this potential, VEGFR-2-CD (the protein tyrosine kinase catalytic domain of VEGFR-2) was cloned and expressed in Streptomyces lividans TK24, a prokaryotic expression system. The recombinant protein was purified, and correlations between its activity and enzyme concentration, ATP concentration, substrate concentration and bivalent cations were characterized. An ELISA-based screening system was then established and used to search for inhibitors acting on the tyrosine kinase part of VEGFR-2. More than 600 compounds originating from a variety of microbes have been screened so far, and a number of them have been demonstrated to be potential inhibitors of VEGFR-2 TK.