Construction of chimeric CblN/Grb2 and its expression in prokaryon / 解放军医学杂志

ABSTRACT

Objective To construct the pGEX-4T-2-CblN/Grb2, express and purify the corresponding chimeric protein; To investigate whether the chimeric CblN/Grb2 possesses ubiquitin ligase activity. Methods Total RNA of SK-BR-3 cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Grb2 SH2 by PCR. The gene fragment encoding the N-terminal part of Cbl protein (named as CblN) was amplified by PCR using pEFHACbl plasmid encoding human Cbl as templates. BamH I and EcoR V restriction enzyme digestion sites were introduced into both flanks of SH2 by overlapping extension PCR and the modified gene were cloned into pcDNA3.1(+). The pcDNA3.1(+)-CblN/Grb2 was obtained by replacing SH2 of CblN with Grb2SH2 and then used as templates to amplify CblN/Grb2 by PCR. The pGEX-4T-2-CblN/Grb2 was constructed by subcloning CblN/Grb2 into the prokaryotic expressing vector pGEX-4T-2. The GST-CblN/Grb2 fusion protein was expressed in E. coli of DH5? under IPTG induction and further purified with Glutathione Sepharose 4B. In vitro ubiquitination assay was performed to investigate whether the GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction, namely whether it possesses ubiquitin ligase activity. Results The fusion expressing vector of pGEX-4T-2-CblN/Grb2 was successfully constructed; The GST-CblN/Grb2 fusion protein was correctly expressed and purified; In vitro ubiquitination assay indicated that GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction and therefore possesses ubiquitin ligase activity. Conclusion Expression, purification of GST-CblN/Grb2 and identification of its′ activity have laid the foundation for further study of chimeric ubiquitin ligase′ effects on the growth of HER2 positive tumor cells.