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

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phosphoregulation in both N- and C-termini. We show that phosphomimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1D19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large Cterminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development

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&gt

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&gt

Purification of MFSD2A 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&gt

Purification of SLC12A5 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&gt

Purification of SLC46A3 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&gt

Purification of SLC12A9 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&gt

Purification of SLC1A1 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&gt

Purification of SLC45A4 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&gt