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Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies

从HEK293F细胞中人K-Cl转运蛋白KCC1的表达和纯化用于结构研究

SL Si Liu
JG Jiangtao Guo

发布: 2021年04月05日第11卷第7期 DOI: 10.21769/BioProtoc.3966 浏览次数: 2747

评审: Chen FanArif Md. Rashedul KabirAkira Karasawa

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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- 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 is 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 for functional and structural studies.

Keywords: KCC (K-Cl转运蛋白) search Mammalian cells (哺乳动物细胞) search Purification (纯化) search Bac-to-Bac baculovirus expression system (杆状病毒表达系统) search BacMam (BacMam) search Structural studies (结构研究) search

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 involve 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. 96-well plate

  2. 6-well plate (Corning, catalog number: 3516 )

  3. Filter systems (250 ml, 0.22 μm; Corning, catalog number: 430767 )

  4. Syringe filters (33 mm, 0.22 µm; Millipore, catalog number: SLGPR33RB )

  5. Amicon Ultra-4 centrifugal filters 100,000 MWCO (Millipore, catalog number: UFC810096 )

  6. pEZT-BM vector (Addgene, catalog number: 74099 )

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

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

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

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

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

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

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

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

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

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

  17. N-dodecyl-β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310S )

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

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

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

  21. D-desthiobiotin (Sigma-Aldrich, catalog number: D1411 )

  22. Tris base (Sangon Biotech, catalog number: A610195 )

  23. KCl (Sangon Biotech, catalog number: A610440 )

  24. PMSF (Sangon Biotech, catalog number: A100754 )

  25. Leupeptin (Sangon Biotech, catalog number: A600580 )

  26. Aprotinin (Sangon Biotech, catalog number: A600153 )

  27. Pepstatin (Sangon Biotech, catalog number: A610583 )

  28. DNase I (Sangon Biotech, catalog number: A610099 )

  29. Resuspension buffer (see Recipes)

  30. Washing buffer (see Recipes)

  31. Elution buffer (see Recipes)

  32. SEC buffer (see Recipes)

  33. Sodium butyrate (2 M) (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air shaker incubator (Zhichu, model: ZCZY-CSV )

  2. 28 °C (shaker) incubator (Zhichu, model: ZCZY-CS9 )

  3. CytoFLEX S Flow Cytometer (Beckman Coulter Life Sciences)

  4. Refrigerated centrifuge (Eppendorf, model: 5424R )

  5. Sonicator (Scientz, model: IID )

  6. High speed centrifuge (Thermo Fisher Scientific, model: Sorvall LYNX 6000 )

  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 One (Thermo Fisher Scientific, catalog number: ND-ONE-W )

Procedure

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文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Liu, S. and Guo, J. (2021). Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies. Bio-protocol 11(7): e3966. DOI: 10.21769/BioProtoc.3966.
  2. Liu, S., Chang, S., Han, B., Xu, L., Zhang, M., Zhao, C., Yang, W., Wang, F., Li, J., Delpire, E., Ye, S., Bai, X. C. and Guo, J. (2019). Cryo-EM structures of the human cation-chloride cotransporter KCC1. Science 366(6464): 505-508.

    3. ris ris Download Citation in RIS Format

分类

生物化学 > 蛋白质 > 分离和纯化

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