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.
Liu
Chunping
C
Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Tian Tan, 100050 Beijing, People's Republic of China.
Guo
Lianhong
L
Yao
Chen
C
Zhang
Ren
R
Li
Yuan
Y
D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*