Plasmid optimization and the localization of the binding site of GPS2-UBC13
Abdullah, Ayesha M.
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The GPS2 protein (G-protein pathway suppressor 2) is a product of the mammalian gps2 gene. It was originally identified and characterized in the context of G protein mitogen-activated protein kinase (MAPK) signaling pathways. Several studies have linked GPS2 with the inhibition of the ubiquitin conjugating enzyme UBC13. GPS2-mediated inhibition of UBC13 regulates several metabolic and inflammatory pathways. It has been shown that a lack of GPS2 is correlated with an increase in adiposity and inflammation due to the aberrant activity of UBC13 affected pathways. Therefore, understanding the relationship between UBC13 and GPS2 will provide further understanding of the molecular processes involved in adipose tissue levels, inflammation and downstream molecular responses. In this study, we attempt to determine the molecular determinants of GPS2 interaction with UBC13 by optimizing the protein expression protocol required to produce GPS2 protein expression in Escherichia coli in quantities viable for biochemical and structural assays. Our results indicate that optimization of the gps2 sequence is required for efficient GPS2 protein expression in E. coli cells. Thanks to this optimization we have been able to successfully express GPS2 full length and several fragments, however, further optimization will be required for assessing GPS2-UBC13 molecular binding via in vitro binding assays.