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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2023 |
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| Enthalten in: |
Journal of visualized experiments : JoVE - (2023), 199 vom: 29. Sept. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Raturi, Sagar [VerfasserIn] |
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| Links: |
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| Themen: |
Journal Article |
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| Anmerkungen: |
Date Completed 01.11.2023 Date Revised 01.11.2023 published: Electronic Citation Status MEDLINE |
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| doi: |
10.3791/65878 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM363360808 |
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| 520 | |a 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 | ||
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| 700 | 1 | |a Li, Huanyu |e verfasserin |4 aut | |
| 700 | 1 | |a Chang, Yung-Ning |e verfasserin |4 aut | |
| 700 | 1 | |a Scacioc, Andreea |e verfasserin |4 aut | |
| 700 | 1 | |a Bohstedt, Tina |e verfasserin |4 aut | |
| 700 | 1 | |a Fernandez-Cid, Alejandra |e verfasserin |4 aut | |
| 700 | 1 | |a Evans, Adam |e verfasserin |4 aut | |
| 700 | 1 | |a Abrusci, Patrizia |e verfasserin |4 aut | |
| 700 | 1 | |a Balakrishnan, Abilasha |e verfasserin |4 aut | |
| 700 | 1 | |a Pascoa, Tomas C |e verfasserin |4 aut | |
| 700 | 1 | |a He, Didi |e verfasserin |4 aut | |
| 700 | 1 | |a Chi, Gamma |e verfasserin |4 aut | |
| 700 | 1 | |a Kaur Singh, Nanki |e verfasserin |4 aut | |
| 700 | 1 | |a Ye, Mingda |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Anna |e verfasserin |4 aut | |
| 700 | 1 | |a Shrestha, Leela |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Dong |e verfasserin |4 aut | |
| 700 | 1 | |a Williams, Eleanor P |e verfasserin |4 aut | |
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| 700 | 1 | |a Ingles-Prieto, Alvaro |e verfasserin |4 aut | |
| 700 | 1 | |a Sauer, David B |e verfasserin |4 aut | |
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