Expression and Purification of the Human Cation-Chloride Cotransporter KCC1 from HEK293F cells for Structural Studies

[Abstract] Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na⁺ and/or K⁺ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl^- driven by the transmembrane K⁺ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins has been challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication (Liu et al., 2019). The procedure may be adapted to functional and structural studies.

Keywords: KCC; Mammalian cells; Purification

[Background] The human Solute Carrier 12 (SLC12) gene family encodes the Cation- Chloride Cotransporters (CCCs) that mediate the electroneutral symport of Cl^- and cations Na⁺ or (and) K⁺ across plasma membranes. Defined by their transport properties and amino acid sequences, CCCs can be divided into several branches, including two Na-K-2Cl cotransporters (NKCC1 and NKCC2), one Na-Cl cotransporter (NCC) and four K-Cl cotransporters (KCC1-KCC4). CCCs play important roles in cell volume regulation, salt reabsorption in kidney, and the GABAergic modulation in neurons. The structural, biochemical and biophysical studies of CCCs involves challenges at the level of protein production and stabilization in the detergent-solubilized state. Baculovirus transduction of HEK293F cells (BacMam) system is a powerful method to heterologously express membrane proteins developed by Eric Gouaux (Goehring et al., 2014). In this protocol, we describe the production of KCC1 with a C-terminal StrepII tag in HEK293F cells using the Baculovirus expression system. The purified protein can be applied for a variety of functional and structural studies.

Materials and Reagents

1. pEZT-BM vector (Addgene, Catalog number: 74099)

2. Sf9 cells (Thermo Fisher Scientific, catalog number: 11496015)

3. HEK293F cells (Thermo Fisher Scientific, catalog number: R79007)

4. DH10Bac Competent Cells (Thermo Fisher Scientific, catalog number: 10361012)

5. 6-well plate (Corning, catalog number: 3516)

6. Penicillin-Streptomycin (Gibco, catalog number: 15140-122)

7. Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106)

8. X-tremeGEN 9 DNA Transfection Reagent (Roche, catalog number: 06365787001)

9. SIM SF Expression Medium (Sino Biological, catalog number: MSF1)

10. SMM 293-TI Expression Medium (Sino Biological, catalog number: M293TI)

11. Sodium butyrate (Sigma-Aldrich, catalog number: 303410)

12. Baculovirus Envelope gp64 Antibody (Thermo Fisher Scientific, catalog number: 12699182)

13. n-dodecyl-β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310S)

14. Cholesteryl Hemisuccinate Tris Salt (CHS) (Anatrace, catalog number: CH210)

15. Glyco-diosgenin (GDN) (Anatrace, catalog number: GDN101)

16. Strep-Tactin Sepharose resin (IBA, catalog number: 2-1201-010)

17. d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411)

18. Amicon Ultra-15 centrifugal filters 100,000 MWCO (Millipore, catalog number: UFC810096)

19. Tris base

20. KCl

21. PMSF

22. Leupeptin

23. Aprotinin

24. Pepstatin

25. DNase I

Equipment

1. 37 °C, 5% CO₂ forced-air shaker incubator

2. 28 °C (shaker) incubator

3. CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences)

4. Refrigerated centrifuge

5. Sonicator

6. High speed centrifuge

7. ÄKTA Purifier chromatography system (GE Healthcare, model: ÄKTA Purifier)

8. SEC column Superose 6 Increase 10/300 GL (GE Healthcare, catalog number: 29- 0915-96)

9. Nanodrop (Thermo Fisher Scientific)

Procedure

A. Cell culture conditions

1. Sf9 cells were cultivated in SIM SF Expression Medium containing 2% fetal bovine serum in shaker incubator at 28 °C, 130 rpm.

2. HEK293F cells were cultivated in SMM 293-TI Expression Medium containing 2% fetal bovine serum in shaker incubator at 37 °C, 130 rpm in the presence of 5% CO₂.

B. Expression of KCC1

1. The full-length human SLC12A4 gene (NCBI accession NP_005063) containing a C-terminal Strep tag was cloned into a pEZT-BM vector.

2. Plasmid was transformed to DH10Bac E. coli to generate the recombinant bacmid.

3. Isolation of bacmid DNA, transfection of Sf9 cells and amplification of virus were following methods from the Bac-to-Bac system (Invitrogen).

4. Measure the baculoviral titer for P2 stocks using flow cytometry with baculovirus envelope gp64 antibody.

5. The baculovirus was used to infect HEK293F cells at a ratio of multiplicity of infection (MOI) 20.

6. HEK293F cells were supplemented with 10 mM sodium butyrate to boost protein expression in 8~12 hours.

7. Cells were cultured in suspension at 37 °C for 48 hours.

8. Harvest the cells by centrifugation at 4000 rpm for 15 min.

C. Purification of KCC1

1. The cell pellet was re-suspended in resuspension buffer (see Recipes) and homogenized by sonication on ice.

2. Add 2% (w:v) n-Dodecyl-β-D-Maltopyranoside (DDM) supplemented with 0.2% (w:v) cholesteryl hemisuccinate (CHS), shake on ice for 2 h to extract membrane proteins.

3. Centrifugation at 18000 rpm for 30 min to remove the pellet.

4. The supernatant was incubated with Strep-Tactin Sepharose resin (IBA) with gentle agitation for 1 hour.

5. The resin was collected on a disposable gravity column, washed in washing buffer (see Recipes) for 10 column volumes.

6. KCC1 was eluted with elution buffer (see Recipes).

7. Load the sample into a Superose 6 Increase 10/300 GL column pre-equilibrated with 1.2 column volume of SEC buffer (see Recipes), at a flow rate of 0.5 ml/min at 4 °C, collecting 0.5 ml fractions.

8. Pool the fractions containing target protein and concentrate the sample to 2 to 7 mg/ml for structural analysis.

Recipes

1. Resuspension buffer

20 mM Tris pH 8.0 150mM KCl

0.1 mM PMSF

2 μg ml⁻¹ DNase I

0.5 μg ml⁻¹ pepstatin 2 μg ml⁻¹ leupeptin 1 μg ml⁻¹ aprotinin

2. Washing buffer

20 mM Tris pH 8.0

150 mM KCl

0.1% (w/v) n-dodecyl-β-D-maltopyranoside 0.02% (w/v) cholesteryl hemisuccinate

3. Elution buffer

20 mM Tris pH 8.0

150 mM KCl

0.06% glyco-diosgenin (GDN)

10 mM d-desthiobiotin

4. SEC buffer

20 mM Tris pH 8.0

150 mM KCl

0.06% glyco-diosgenin (GDN)

Acknowledgments

This work was supported in part by Ministry of Science and Technology (2018YFA0508100), the National Natural Science Foundation of China (31870724), and Zhejiang Provincial Natural Science Foundation (LR19C050002).

References

1. Liu, S., S. Chang, B. Han, L. Xu, M. Zhang, C. Zhao, W. Yang, F. Wang, J. Li, E. Delpire, S. Ye, X. C.Bai & J. Guo (2019) Cryo-EM structures of the human cation- chloride cotransporter KCC1. Science 366(6464): 505-508.

2. Goehring, A., C. H. Lee, K. H. Wang, J. C. Michel, D. P. Claxton, I. Baconguis, T. Althoff, S. Fischer, K. C. Garcia & E. Gouaux (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9(11): 2574-85.

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