Cloning, Expression and Characterization of
Human Vascular Endothelial Growth Factor Receptor 1 Tyrosine Kinase
ZHUANG Shu-Fei, YE Qi-Zhuang*
( National Center for Drug Screening,
Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract Vascular
endothelial growth factor receptor 1 (Flt1) plays an important role in
angiogenesis. It was hypothesized that, upon binding to VEGF, Flt1tyrosine
kinase underwent dimerization and initiated the signal transduction in
VEGF/VEGF receptor system. In this report, a soluble active Flt1 tyrosine
kinase domain expressed in E.coli was obtained, and its properties were
partly characterized. The cDNA of Flt1 tyrosine kinase domain was obtained from
the total RNA extracted from human liver cancer tissues by using RT-PCR, and
was cloned to vector pGEX-KG. A soluble active GST-fusion protein of Flt1
tyrosine kinase domain (GST-F) was obtained from E.coli BL21 (DE3)
pLysS. Athough it was reported that GST-F contains no phosphorylation site, it
did autophosphorylate in vitro. Mg2+ and Mn2+ were
essential for the activity. It was also found that GST-F phosphorylated a
synthesized substrate PolyE4Y, but not MBP and Src-related-peptide. The optimal
Mg2+ and Mn2+ concentration for polyE4Y phosphorylation
was 15 mmol/L and 0.5 mmol/L, respectively. This work is helpful for developing
the new anti-cancer drugs.
Key words vascular endothelial growth factor receptor;
tyrosine kinase; fusion protein
*Corresponding author: Tel, 86-21-50800721; Fax, 86-21-50800721;
e-mail, [email protected]