Expression for the phosphate translocator from Toxoplasma gondii in Escherichia coli and purification of the recombinant protein
Plastids are plant specific cell organelles, which perform many specialized and essential functions. Integration of plastids into cellular metabolism requires an extensive exchange of metabolites between plastids and the surrounding cytosol which is catalyzed by more than 150 different transporters. One of the best-characterized families is the plastid phosphate translocator family (pPT). Proteins similar to plant pPTs (APTs) where recently identified in Apicomplexa, e.g. in Plasmodium falciparum and Toxoplasma gondii. Parasitic diseases caused by these species are one of the most serious medical and economic issues worldwide. The APTs show similar activities and substrate specificities as their plant counterparts. Most importantly, the disruption of the APT gene in T. gondii leads to immediate death of the parasite which clearly shows the importance of this transporter for growth of the parasites. Therefore, there is an ongoing project to elucidate the structure of the APT from T. gondii at atomic resolution and to analyze the transport mechanism at a molecular level by x-ray crystallography. To achieve that goal, the APT protein has to be expressed in a heterologous system. In this study the apicoplast phosphate transporter protein was produced by heterologous expression in Escherichia coli. The suitable plasmid (pET21b) was selected, covering the TgAPT gene, introduced into BL21 RIL strain of E. coli and the recombinant machinery was induced with IPTG. The APT protein was engineered with a Twin-Strep®-affinity tag attached to its N-terminus and was purified by affinity chromatography on Strep-Tactin® resins. The successful expression of the APT in E. coli offers the possibility to produce and purify the APT protein in larger quantities, and, second, to measure its transport activity directly in the bacterial cells.
ForlagUiT Norges arktiske universitet
UiT The Arctic University of Norway
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