(116am) Protein CO-Expression of Human Centrin 2 and Human SFI1 Binding Domains

Department of Chemistry, University of Puerto Rico, Mayaguez, PR

Human Centrin 2 (HCen2) and Human Sfi1 (hSfi1) are vital proteins involved in proper centriole duplication and separation. These proteins are also found as components of contractile fibers that make up microtubules to ensure appropriate cell division. The objective of this research is to co-express these two proteins as a soluble bound complex for purification and future protein interaction studies. HCen2 and several hSfi1 binding domains were cloned into a pGEX Dicistronic co-expression vector containing two ribosomal binding sites. The benefits of co-expression include the acquisition of a highly soluble protein complex that can be easily purified. The cloned vector was then transformed into BL21(DE3) Star E.coli bacterial strain cells. The cells were grown following a low scale expression protocol. UV spectroscopy was used to determine cell optical density, and finally protein expression was induced with IPTG at log phase. Growth cell pellets were conserved and SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to confirm protein expression. This low- scale expression will provide the necessary data to proceed with a large scale fermentor based expression and purification.