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Biotechnol. Appl. Biochem. (2008) 50, (113–119) (Printed in Great Britain)
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| Expression and purification of human vascular-endothelial-growth-factor-receptor-2 tyrosine kinase in Streptomyces for inhibitor screening |
| Chunping Liu*, Lianhong Guo*, Chen Yao*, Ren Zhang*† and Yuan Li*1 |
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| *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, and †School of Biological Sciences, University of Wollongong, Northfields Ave., Wollongong, NSW 2522, Australia
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Key words: ELISA, inhibitor, receptor tyrosine kinase, Streptomyces, vascular endothelial growth factor (VEGF), vascular-endothelial-growth-factor-receptor 2 (VEGFR-2).
Abbreviations used: AP, alkaline phosphatase; CD, catalytic domain; H+L, heavy and light chains; HUVEC, human umbilical-vein endothelial cell; RT, reverse transcription; TBS, Tris-buffered saline; TK, tyrosine kinase; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
1To whom correspondence should be addressed (email yuanwli@263.net).
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.
Received 14 May 2007/2 September 2007; accepted 10 September 2007
Published as Immediate Publication 10 September 2007, doi:10.1042/BA20070112
© 2008 Portland Press Ltd
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