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- W3004822432 abstract "The human Na+-coupled citrate transporter (SLC13A5) assists in the uptake of sodium ions and citrate3- ion into the cell. This plasma membrane cotransporter is expressed in brain, liver and testis cells. In the brain, SLC13A5 is mostly expressed in neurons, where citrate might help as energy source in starvation or uncontrolled diabetes. Furthermore, loss-of-function mutations in SLC13A5 are associated with early infantile epileptic encephalopathy. Children with this rare disease develop seizures after birth and throughout the rest of their life. They show speech difficulty, and slow and limited motor skills. Also, some studies suggest that the transporter can be related to Alzheimer's disease. The purpose of this research is to express and purify human SLC13A5 for biochemical and biophysical studies. Yeast Pichia Pastoris is a well-established protein expression system that produces high cell density growth at affordable cost. Moreover, different eukaryotic membrane proteins showed successful expression in this system. To facilitate cellular localization and purification N-terminal and C-terminal tags were engineered and tested to support transport function. An N-terminal His10-SUMO domain was tested for its ability to enhance solubility and protein folding. A C-terminal GFP -Twin-Strep-tag was engineered to facilitate cellular localization studies and protein purification. Fluorescence microscopy showed His10-SUMO- SLC13A5-GFP-Strep localized to the endoplasmic reticulum and plasma membrane in P. pastoris cells. Using [14C]-citrate, we demonstrate Na+-coupled citrate transport in cells. Thus, P. pastoris may provide excellent source material for future protein purification." @default.
- W3004822432 created "2020-02-14" @default.
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- W3004822432 date "2020-02-01" @default.
- W3004822432 modified "2023-10-18" @default.
- W3004822432 title "Expression of Functional Human Na+- Coupled Citrate Transporter (SLC13A5) in the Yeast Pichia pastoris" @default.
- W3004822432 doi "https://doi.org/10.1016/j.bpj.2019.11.1250" @default.
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