30 research outputs found
Structural characterization of human urea transporters UT-A and UT-B and their inhibition
In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters
High-throughput expression and purification of human solute carriers for structural and biochemical studies
Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening
Western Star (Corner Brook, N.L.), 1911-12-20
The Western Star began publication on Newfoundland's west coast on 4 April 1900, appearing weekly with brief semiweekly periods up to 1952, when it became a daily. The current collection contains 21 April 1900 - 31 March 1926
Purification of SLC31A1 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC15A4 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC25A13 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC7A1 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC12A6 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC17A5 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>