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Oct 2019

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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   

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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), Mammalian cells (哺乳动物细胞), Purification (纯化), Bac-to-Bac baculovirus expression system (杆状病毒表达系统), BacMam (BacMam), Structural studies (结构研究)

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

  1. Cell culture conditions

    1. Sf9 cells were cultivated in SIM SF Expression Medium containing 2% fetal bovine serum and 0.5% Penicillin-Streptomycin in shaker incubator at 28 °C, 130 rpm. Maintain the suspension Sf9 cells between 0.5 × 106-4 × 106 cells/ml.

    2. HEK293F cells were cultivated in SMM 293-TI Expression Medium containing 2% fetal bovine serum and 0.5% Penicillin-Streptomycin in shaker incubator at 37 °C, 130 rpm in the presence of 5% CO2. Maintain the suspension HEK293F cells between 0.5 × 106-4 × 106 cells/ml. It is best to passage the cells by no more than two months.


  2. Expression of KCC1

    1. The full-length human SLC12A4 gene (NCBI accession NP_005063) and a C-terminal Strep tag connected by a GGSSGG linker were cloned into a pEZT-BM vector.

    2. 500 ng plasmid was added to 100 μl DH10Bac E. coli to Generate the recombinant bacmid.

    3. Isolation of bacmid DNA (page 20-22), transfection of Sf9 cells (page 25-28) and amplification of virus (page 30-31) were following methods from the Bac-to-Bac system (Invitrogen) (Reference 1).

    4. Measure the baculoviral titer for P2 stocks using flow cytometry with baculovirus envelope gp64 antibody (Figure 1). Briefly,

      1. In 96-well block, add 100 μl Sf9 cells/well (cell density = 2 × 106 cells/ml).

      2. Dilute virus stock 10× in an Eppendorf tube.

      3. In a 96-well plate, set up serial dilution of 10× diluted stock:

        1. 250× (4 μl 10× + 96 μl media)

        2. 500× (50 μl 250× + 50 μl media)

        3. 1,000× (50 μl 500× + 50 μl media)

        4. 2,000× (50 μl 1,000× + 50 μl media)

      4. Transfer 20 μl of serially diluted samples from 96-well plate to 96-well block.

      5. Seal block and place in shaker for 18 h at 28 °C and 200 rpm.

      6. Stain Sf9 with anti-gp64-PE conjugated antibody for 20 min in the dark and analyze cell count by flow cytometry.

      7. For infected values above 30%: IU = Total cell number × [-ln (1 - %infected value/100)] × [viral dilution factor]/(volume of inoculum); for infected values below 30% (more reliable): IU = Total cell number × (%infected value/100) × [viral dilution factor]/(volume of inoculum).



      Figure 1. Estimation of baculoviral titer using flow cytometry. Histogram plots of cell count as a function of PE signal, indicating distinct populations for (A) uninfected Sf9 cells, as well as (B) PE-positive infected Sf9 cells.


    5. The baculovirus was used to infect HEK293F cells of a density of 2 × 106-3.5 × 106 cells/ml 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 h.

    7. Cells were cultured in suspension at 37 °C and 130 rpm for 48 h.

    8. Harvest the cells by centrifugation at 4,600 × g for 15 min.


  3. Purification of KCC1

    1. The cell pellet was re-suspended in 15-20 ml per liter of expression culture resuspension buffer and homogenized by sonication (2 s sonication and 5 s of pause in 40 cycles, 300 kW) 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. Centrifuge at 38,000 × g for 30 min to remove the pellet.

    4. The supernatant was incubated with 2 ml per liter of expression culture Strep-Tactin Sepharose resin (IBA) with gentle agitation for 1 h.

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

    6. KCC1 was eluted with elution buffer. Analyze on SDS-PAGE gel (Figure 2). Typical yield at this step is ~2-3 mg per liter of expression culture.



      Figure 2. 12% SDS-PAGE analysis of purified KCC1 stained with Coomassie Brilliant Blue


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

    8. Pool the fractions based on the gel filtration chromatogram (Figure 3) and concentrate to 2 to 7 mg/ml using an Amicon Ultra-4 centrifugal filters (100 kDa cut-off) for structural analysis.



      Figure 3. Purification of KCC1 by size-exclusion chromatography. The purified KCC1 is monodisperse.

Recipes

  1. Resuspension buffer

    20 mM Tris (pH 8.0)

    150 mM KCl

    0.1 mM PMSF

    2 μg/ml DNase I

    0.5 μg/ml pepstatin

    2 μg/ml leupeptin

    1 μg/ml aprotinin

    Chill the buffer to 4 °C. Add DNase I immediately before use, PMSF, Aprotinin, Leupeptin and Pepstatin A

  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

    Store at 4 °C for up to 1 week

  3. Elution buffer

    20 mM Tris (pH 8.0)

    150 mM KCl

    0.06% glyco-diosgenin (GDN)

    10 mM d-desthiobiotin

    Store at 4 °C for up to 1 week

  4. SEC buffer

    20 mM Tris (pH 8.0)

    150 mM KCl

    0.06% glyco-diosgenin (GDN)

    Filter using a 0.22 μm filter

    Store at 4 °C for up to 1 week

  5. Sodium butyrate (2 M)

    Dissolve 11 g of sodium butyrate with water to a final volume of 50 ml and filter-sterilize using a 0.22 μm filter inside the biological safety cabinet. Store at -20 °C for at least 1 month

Acknowledgments

This work was supported in part by Ministry of Science and Technology (2018YFA0508100), the National Natural Science Foundation of China (31870724 and 32000853), and Zhejiang Provincial Natural Science Foundation (LR19C050002).

Competing interests

There are no conflicts of interest or competing interest.

References

  1. Bac-to-Bac Baculovirus Expression System User Guide. https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0000414_BactoBacExpressionSystem_UG.pdf&title=VXNlciBHdWlkZTogQmFjLXRvLUJhYyBCYWN1bG92aXJ1cyBFeHByZXNzaW9uIFN5c3RlbQ==
  2. Goehring, A., Lee, C. H., Wang, K. H., Michel, J. C., Claxton, D. P., Baconguis, I., Althoff, T., Fischer, S., Garcia, K. C. and Gouaux, E. (2014). Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.Nat Protoc 9(11): 2574-2585.
  3. 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.

简介

[摘要]阳离子-氯化物共转运蛋白(CCC)介导诸如Na +和/或K +的阳离子与氯离子在膜上的耦合,电中性共价。间幼儿中心家庭,K-CL协同转运蛋白(KCC1-KCC4)抽UDE细胞内氯-通过跨膜ķ +梯度。在人类中,这些KCC在神经系统和肾脏的生理中起着至关重要的作用。然而,特定的KCC性质保持基本机制知之甚少,部分是因为膜蛋白的纯化是具有挑战性的。在这里,我们介绍了从我们最近的出版物中使用的HEK293F细胞中纯化全长KCC1的方案(Liu等人,2019)。该程序可适用于功能和结构研究。

[背景]人类溶质载体12(SLC12 )基因家族编码阳离子的氯化物协同转运蛋白(CCCS)介导Cl组成的电中性同向转运-和阳离子的Na +或(和)K +跨越质膜。根据其转运特性和氨基酸序列定义,CCC可分为几个分支,包括两个Na-K-2Cl协同转运蛋白(NKCC1和NKCC2),一个Na-Cl协同转运蛋白(NCC)和四个K-Cl协同转运蛋白(KCC1-KCC4 )。CCC在细胞体积调节,肾脏盐分重吸收和神经元GABA能调节中起重要作用。CCC的结构,生化和生物物理研究涉及在去污剂溶解状态下蛋白质生产和稳定方面的挑战。杆状病毒转导HEK293F细胞(BacMam)系统是异源表达由Eric Gouaux开发的膜蛋白的有效方法(Goehring等人,2014)。在此协议中,我们描述了使用杆状病毒表达系统在HEK293F细胞中带有C末端StrepII标签的KCC1的生产。纯化的蛋白质可用于各种功能和结构研究。

关键字:K-Cl转运蛋白, 哺乳动物细胞, 纯化, 杆状病毒表达系统, BacMam, 结构研究

材料和试剂
1. 96孔板     

2. 6孔板(Corning,目录号:3516)     

3.过滤系统(250毫升,0.22微米;康宁,目录号:430767)     

4.注射器过滤器(33 mm,0.22 µm ;Millipore,目录号:SLGPR33RB)     

5. Amicon Ultra-4离心过滤器100,000 MWCO(密理博,目录号:UFC810096)     

6. pEZT-BM载体(Addgene公司,Ç atalog号码:74099)     

7. Sf9细胞(Thermo Fisher Scientific,目录号:11496015)     

8. HEK293F细胞(Thermo Fisher Scientific,目录号:R79007)     

9. DH10Bac感受态细胞(Thermo Fisher Scientific,目录号:10361012)     

10.青霉素-链霉素(Gibco,目录号:15140-122) 

11.胎牛血清(FBS)(Gibco,目录号:10270-106) 

12. X-tremeGEN 9 DNA转染试剂(罗氏,目录号:06365787001) 

13. SIM SF表达培养基(中生物,目录号:MSF1) 

14. SMM 293-TI表达培养基(Sino Biological,目录号:M293TI) 

15.丁酸钠(Sigma-Aldrich,目录号:303410) 

16.杆状病毒包膜gp64抗体(Thermo Fisher Scientific,目录号:12699182) 

17. N-十二烷基-β-D-麦芽吡喃糖苷(DDM)(Anatrace,目录号:D310S) 

18.胆固醇半琥珀酸酯三盐(CHS)(Anatrace,目录号:CH210) 

19.糖原-薯s皂苷元(GDN)(Anatrace,目录号:GDN101) 

20. Strep-Tactin Sepharose树脂(IBA,目录号:2-1201-010) 

21. DD esthiobiotin(Sigma-Aldrich公司,目录号:D1411) 

22. Tris base(Sangon Biotech,目录号:A610195) 

23. KCl(Sangon Biotech,目录号:A610440) 

24. PMSF(Sangon Biotech,目录号:A100754) 

25. Leupeptin(Sangon Biotech,目录号:A600580) 

26.抑肽酶(Sangon Biotech,目录号:A600153) 

27.抑肽酶(Pepstatin)(Sangon Biotech,目录号:A610583) 

28.脱氧核糖核酸酶I (Sangon Biotech,目录号:A610099) 

29.重悬缓冲区(请参阅食谱) 

30.洗涤缓冲液(请参见食谱) 

31.洗脱缓冲液(请参见配方) 

32. SEC缓冲区(请参阅食谱) 

33.丁酸钠(2 M)(请参阅食谱) 



设备
37 °C,5%C O 2强制空气振荡器培养箱(Zhichu,型号:ZCZY-CSV)
28 °C(摇床)恒温箱(Zhichu,型号:ZCZY-CS9)
CytoFLEX S流式细胞仪(贝克曼库尔特生命科学公司)
冷冻离心机(Eppendorf,型号:5424 R)             
Sonicator(Scientz,型号:IID)
高速离心机(Thermo Fisher Scientific,型号:Sorvall LYNX 6000)
ÄKTAPurifier色谱系统(GE Healthcare,型号:ÄKTAPurifier)
SEC柱Superose 6增加10/300 GL(GE Healthcare,目录号:29-0915-96)
Nanodrop One(Thermo Fisher Scientific ,目录号:ND-ONE-W)


程序
A.细胞培养条件     

将Sf9细胞在28°C,130 rpm的摇床培养箱中的SIM SF表达培养基中培养,该培养基含有2%的胎牛血清和0.5%的Penicil lin-链霉素。保持悬液Sf9细胞在0.5 ×10 6 -4 ×10 6细胞/ ml之间。
将HEK293F细胞在含有2%胎牛血清和0.5%青霉素-链霉素的SMM 293-TI表达培养基中于saker培养箱中于37°C,130 rpm,5%CO 2的条件下培养。保持悬浮HEK293F细胞在0.5 ×10 6 -4×10 6细胞/ ml之间。最好让细胞传代不超过2个月。


B. KCC1的表达     

全长人SLC12A4基因(NCBI登录NP_005063)和C-末端标签链球菌连接ED通过GGSSGG接头克隆入pEZT-BM矢量。
将500ng质粒添加到100μlDH10Bac大肠杆菌中以产生重组杆粒。
的隔离杆粒DNA(20-22页),Sf9细胞的转染(25-28页)和病毒的扩增(30-31页)的下列方法从上述BAC到北系统(Invitrogen) (参考文献1) 。
使用带有杆状病毒包膜gp64抗体的流式细胞仪测量杆状病毒滴度,以检测P2储备(图1)。简要地,
在96 -孔块,添加100μl的Sf9细胞/孔(细胞密度= 2 × 10 6细胞/ ml)。
在eppendorf管中稀释10倍病毒。
在96 -孔平板上,建立的10系列稀释×稀释的储备:
250 × (4μl10 × + 96μl介质)
500 × (50μl250 × + 50μl介质)
1 ,000 × (50微升500 × + 50微升培养基)
2 ,000 × (50微升1 ,000 × + 50微升培养基)
转移20微升从96系列稀释的样品的-孔板96 -孔阻塞。
密封并在28 ° C和200 rpm的振动器中放置18小时。
在黑暗中用抗gp64-PE共轭抗体将Sf9染色20分钟,并通过流式细胞仪分析细胞计数。
对于大于30%的感染值:IU =总细胞数× [-ln(1--感染率%/ 100)] × [病毒稀释因子] /(接种量);如果感染值低于30%(更可靠):IU =总细胞数× (感染率%/ 100)× [病毒稀释因子] /(接种量)。

图1.使用流式细胞仪估算杆状病毒滴度。细胞计数作为PE信号的函数的直方图,indicat荷兰国际集团不同的种群为(A)未感染的Sf9细胞,以及(B)PE-阳性感染的Sf9细胞。



的杆状病毒被用于感染HE 2的密度的K293F细胞×10 6 - 3.5×10 6细胞毫升/在感染复数(MOI)20的多重性的比率。
HEK293F细胞补充有10 mM丁酸钠,以在8-12小时内增强蛋白质表达。
将细胞在悬浮液中于37 ° C和130 rpm培养48小时。
以4,600 × g离心15分钟收获细胞。


C.纯化KCC1的     

将细胞沉淀重悬于每升表达培养物重悬缓冲液15-20 ml中,并在冰上通过超声处理(2 s超声处理和5 s暂停,40个循环,300 kW)进行匀浆。
加入2%(w:v)正十二烷基-β-D-麦芽吡喃糖苷(DDM),补充0.2%(w:v)胆固醇半琥珀酸酯(CHS),在冰上振摇2小时以提取膜蛋白。
以38,000 × g离心30分钟以除去沉淀物。
将上清液与2 ml / l的表达培养物S trep-Tactin Sepharose树脂(IBA)轻轻搅动孵育1小时。
将树脂收集在一次性重力柱上,在洗涤缓冲液中洗涤10倍柱体积。
用洗脱缓冲液洗脱KCC1。在SDS-PAGE凝胶上进行分析(图2)。该步骤的典型产量为每升表达培养物约2-3 mg 。

图2.用考马斯亮蓝染色的纯化KCC1的12%SDS-PAGE分析

将样品加载到用1.2柱体积的SEC缓冲液预平衡的Superose 6增加10/300 GL色谱柱中,在4°C下以0.5 ml / min的流速收集0.5 ml馏分。
根据凝胶过滤色谱图(图3)合并级分,并使用Amicon Ultra-4离心过滤器(截止值为100 kDa)浓缩至2至7 mg / ml或进行结构分析。

图3.通过大小排阻色谱法纯化KCC1。纯化的KCC1是单分散的。

菜谱
重悬缓冲
20 mM Tris (pH 8.0 )
150毫米氯化钾
0.1 mM的PMSF
2μg / ml脱氧核糖核酸酶I
0.5μg / ml胃抑素
2μg / ml亮肽素
1μg / ml抑肽酶

将缓冲液冷却至4 °C。一个DD的DNase I我mmediately使用前,PMSF,抑肽酶,亮肽素和胃蛋白酶抑制剂A


洗涤缓冲液
20 mM Tris (pH 8.0 )


150毫米氯化钾


0.1%(w / v)正十二烷基-β-D-麦芽吡喃糖苷


0.02%(w / v)胆固醇半琥珀酸酯


储存在4°C下长达1周


洗脱缓冲液
20 mM Tris (pH 8.0 )


150毫米氯化钾


0.06%糖原-薯gen皂苷元(GDN)


10 mM d-脱硫生物素


储存在4°C下长达1周


SEC缓冲区
20 mM Tris (pH 8.0 )


150毫米氯化钾


0.06%糖原-薯gen皂苷元(GDN)


使用0.22μm过滤器过滤


储存在4°C下长达1周


丁酸钠(2 M)
用水溶解11克丁酸钠至最终体积为50毫升,并在生物安全柜内使用0.22μm过滤器进行过滤灭菌。储存在- 20℃下至少1个月



致谢



这项工作得到了科学技术部(2018YFA0508100),国家自然科学基金(31870724和32000853 )和浙江省自然科学基金(LR19C050002)的部分支持。


利益争夺



没有利益冲突或利益冲突。



参考



Bac-to-Bac杆状病毒表达系统用户指南。https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0000414_BactoBacExpressionSystem_UG.pdf&title=VXNlciBHdWlkZYB1HXNY1R9YR1NR1YG1YR1YR1YR1YR1N1R1B1D9B9B1D0C
Goehring ,A.,Lee,CH,Wang,KH,Michel,JC,Claxton,DP,Baconguis,I.,Althoff,T.,Fischer,S.,Garcia,KC和Gouaux,E. (2014)。膜蛋白在哺乳动物细胞中的筛选和大规模表达,用于结构研究。Nat Protoc 9(11):2574-2585。
刘珊,张珊,韩冰,徐琳,张明,赵成,杨威,王芳,李健,德尔皮尔Ye,S.,Bai,XC and Guo,J.(2019年)。人类阳离子-氯化物共转运蛋白KCC1的Cryo-EM结构。科学366(6464):505-508。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
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.
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