[Cloning, expression and characterization of human vascular endothelial growth factor receptor 1 tyrosine kinase].
Vascular endothelial growth factor receptor 1 (Flt1) plays an important role in angiogenesis. It was hypothesized that, upon binding to VEGF, Flt1 tyrosine 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. Although 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.
Zhuang
Shu-Fei
SF
National Center for Drug Screening, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031.
Ye
Qi-Zhuang
QZ
D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D040281:Vascular Endothelial Growth Factor Receptor-1
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D008345:Manganese; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D008274:Magnesium