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, ...

University of Oxford

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning.

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Screening and Production of Recombinant Human Proteins: Protein Production in E. coli.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.

Screening and Production of Recombinant Human Proteins: Protein Production in Insect Cells.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.

Expression Screening of Human Integral Membrane Proteins Using BacMam.

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 phospho-regulation in both N- and C-termini. We show that phospho-mimetic 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, KCC1Δ19, 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 C-terminal 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.

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

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.

Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

Alejandra Fernandez-Cid

Centre for Medicines Discovery,
Nuffield Department of Medicine,
Centre for Medicines Discovery, Nuffield Department of Medicine

University of Oxford

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Alejandra Fernandez-Cid

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University of Oxford

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Centre for Medicines Discovery,
Nuffield Department of Medicine,
Centre for Medicines Discovery, Nuffield Department of Medicine