A. thaliana G protein y-subunit gene : cloning, characterization and expression

Official URL: http://risc01.sabanciuniv.edu/record=b1082170 (Table of Contents)

The aim of this thesis was to characterize, clone and express AGG1 gene product for structural studies either on its or own together with beta and alpha subunits. The AGG1 gene coding for the Arabidopsis thaliana G protein gamma subunit was amplified by PCR and subcloned in E. coli for verification and analyses of the cDNA sequence. Following source sequence verification AGG1 was inserted into different expression vector for overexpression of the recombinant protein. These vectors included pGEX-4T2, pGFPuv, pTrcHis-TOPO and pT7/NT-TOPO. Different E. coli strains including BL21(DE3) and BL21(DE3)pLysS, were tried as host cells. Expression of AGG1-his tag fusion protein using pTrcHis-TOPO in BL21(DE3)pLysS cells was demonstrated. AGG1 protein was detected by Western blotting and coomassie blue staining of polyacrylamide gels. This study is the first report of AGG1 expression and synthesis of the gene product in a bacterial cell and it provides characterization of different AGG1 gene containing constructs. Further work is needed for more efficient expression of AGG1 in other host systems.