Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E-coli

Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiatio n and are involved in signal transduction. Uncontrolled signaling from rece ptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamm atory responses and diseases such as cancer and atherosclerosis. Thus, inhi bitors that block the activity of tyrosine kinases or the signaling pathway s of PTKs activation could be assumed as the potential candidate for drug d evelopment. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK g ene was amplified by PCR using the cDNA of ab1 from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tag ged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5 alpha and was induced to express PTK protein. The exp ression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reach ed peak level about 40% of the host total protein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PT K fusion protein presented higher activity for tyrosine phosphorylation.