Expression of Src Homology 2 Domain of v-yes Oncogene in E.coli

다국어 초록 (Multilingual Abstract)

To investigate the function of the Src Homology 2 (SH2) domains found in various non-receptor protein tyrosine kinases (PTKs) and many other cytoplasmic signalling proteins, the partial sequence of the v-yes oncogene containing the SH2 domain has been expressed in E. coli as a fused protein. The 456 by Sau3AI-HaeIII fragment of v-yes was inserted into BamHI-SmaI site of the pGEX-2T vector which has tac promoter and makes the fused proteins with glutathione S-transferase (GST). The fused protein was induced by IPTG and purified from E.coli crude extracts with glutathione-agarose beads. The GST moiety of the fused protein was excised by thrombin treatment. To assay the affinity of the expressed SH2 domain to the tyrosine-phosphorylated proteins, horse raddish peroxidase (HRP)-conjugated SH2 domain was used in detecting phosphotyrosines of enzymatically phosphorylated glutamate-tyrosine copolymers (PGT).