Cloning And Expression Of Ab1 Protein Tyrosine Kinase(PTK) Gene And The Screening Of PTK Inhibitors Form Chinese Traditional Medicinal Herbs

Signal transduction cascades that are driven by tyrosine phosphorylation regulate cell proliferation and differentiation. The initiation, extent and termination of tyrosine phosphorylation are determined by the converted activity of protein tyrosine kinase (PTK) and protein tyrosine phosphorylase (PTPase). PTK and PTPase catalyze a reversible reaction for protein phosphorylation and dephosphorylation, which is named protein reversible phosphorylation (PRP). PRP is a universal regulation pathway which controls the primary physiological and pathological process such as growth and development of cell, gene expression and regulation, synthesis and release of neurotransmitters et al. Furthermore, PRP plays a key role to regulate the central neural system in the signal transduction .As the direct or indirect acting enzymes, PTK and PTPase regulate the activities of other enzymes or proteins via phosphorylation and dephosphorylation which make the cell to response the stimulation of environment signal and maintain the physiological metabolism. Otherwise, the pathological or carcinogenic event will take place.It has been shown that the overexpression of PTK will lead to cell transformation, carcinogenosis and metastasis. This means that if molecules, which inhibit the PTK activities, can be found, they can be used as signal transduction regulators to disrupt the linkage of PTKs overexpression and transformation, and restore the normal regulation of cell growth and differentiation, which possess the potential for tumor prevention and treatment. Therefore, one of the trends for developing anti-cancer drugs is to screen the PTK inhibitors from natural medicine or to synthesize the PTK inhibition compounds. And even, the PTK and the other molecules involving in signal transduction are to be the new targets for drug development.On the assumption mentioned above, an attempt was tried in this paper to clone and express the abl PTK gene to purify the PTK protein used as indicator to screen PTK inhibitors from the extracts of Chinese traditional herbs (CTHs) aimed at developing anticancer natural medicine. The v-abl gene was found in the Abelson murine leukemia virus, which caused leukemia in mice. Abl PTK was a multidomain protein that contained several common regions involved in signal transduction such as SH2, SH3 and tyrosine kinase domains.In this paper, Abl-PTK gene was cloned and expressed in E.coli. Using the purified recombinant PTK as indicator, 7 CTHs, which displayed higher PTK activity inhibition ability, were screened from 30 CTHs. And the effects of 3 selected CTHs on the growth and PTK activities of Hela cell were also investigated.Part I Cloning of Abl-PTK geneTaking the V-abl cDNA of murine leukemia virus as template, the PTK gene, expecting length with 860 bp, was amplified using PCR amplification procedure. After digested with endonuclease BamHl and î–©oRI , the amplified fragment was directional inserted into GST-fusion expression vector pGEX2T. Thus, the recombinant plasmid containing PTK gene, named pGEX-PTK, was constructed. The recombinant plasmid pGEX-PTK was transformed into E.coli DH5a competent cell and the Amp-resistant transformants were screened from LB plates containing Ampencilin (lOOug/mL). The recombinant plasmid was identified using endonuclease digestion assay and DNA sequencing. The result to compare the cloned gene with v-abl-PTK gene registered in GenBank (No.AF033812) showed that the PTK gene was correctly cloned into the vector and frame-in with the GST protein reading frame.Part II Optimization of PTK expression and purification of recombinant PTK protein.The effect of factors affecting PTK expression was investigated and the optimized condition for PTK expression was established. And the recombinant PTK was purified using GST-Sepharose 4B affinity chromatography.1. The time course for PTK induction expression showed that the specific expressed PTK appeared after 10 min induction with IPTG. And it reached the top level for expression...