Cloning and expression of beta subunit (AGB1) of heterotrimeric g-protein complex A.thaliana

Official URL: http://risc01.sabanciuniv.edu/record=b1534429 (Table of Contents)

Heterotrimeric guanine nucleotide-binding proteins(G proteins) act as molecular switches in signaling pathways by coupling the activation of receptors at the cell surface to intracellular responses.The heterotrimeric G protein complex consists of alpha, beta and gamma subunits. G proteins are the most prevalent signaling systems in mammalian cells, playing a role in the regulation of sensory perception, cell growth and hormonal regulation. In plants, G proteins play regulatory roles in multiple developmental processes ranging from seed germination and early seedling development to root development and organ shape determination. Our group is involved in structural studies of G proteins from Arabidopsis thaliana and the work presented in this thesis contributes to the development of purification process of AGB1 protein in E.coli. The aim of this study was to express the A.thaliana beta subunit (AGB1) in E.coli using pMCSG7 vector. In this system the protein is expressed with a his-tag which can be removed after digestion with tobacco etch virus (TEV) protease at the TEV cleavage site introduced from the vector. For cloning AGB1 gene into pMCSG7 vector, ligation-independent cloning (LIC) which allows insertion of DNA fragments independent of restriction sites and ligases was used. The AGB1 gene (in pMCSG7 vector) was expressed in Top10, DH5α and BL21plus* E.coli cells. The optimum protein expression was observed in BL21plus* cells. Efforts focused on optimization of protein expression and on obtaining AGB1 in a pure state by developing a purification protocol involving affinity, ion exchange and size exclusion chromatography. Another aim of this study was to establish the protocols for expression and purification of Tobacco Etch Virus (TEV) protease in our lab in order to cleave the his-tag from AGB1 efficiently in a cost effective way.pMHTDelta238 vector (with TEV protease gene) was introduced intoBL21De3 cells. His tagged TEV protease was expressed and isolated with affinity chromatography. The activity of TEV protease was confirmed using a control protein called BTL2 and western blot analysis. The purified AGB1 protein was cleaved with TEV protease to remove the his-tag. Results were observed by western blot and the efficiency of cleavage with TEV protease was verified by comparing uncleaved and cleaved AGB1 protein samples.