Summary

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Published: December 31, 2019
doi:

Summary

我们描述了一种在大肠杆菌中的异质表达和利用法国印刷机对细胞进行分理的膜囊泡制剂中质子驱动膜输送剂的表征方法。

Abstract

已经开发出几种方法,以在功能上表征新型膜输送机。多胺在所有生物体中普遍存在,但植物中的多胺交换剂尚未确定。在这里,我们概述了一种使用从大肠杆菌细胞的解毒中产生膜囊泡来描述多胺抗结剂的方法,这种细胞异构体表达植物抗结剂。首先,我们在缺乏多胺和精氨酸交换输送机的大肠杆菌菌株中异构地表示AtBAT1。囊泡是使用法国印刷机生产的,通过超离心净化,并用于标记基材的膜过滤测定,以证明输送机的基板特异性。这些测定表明,AtBAT1是精氨酸、β-氨基丁酸(GABA)、普赖辛和精氨酸的质子介导物。为AtBAT1测定而开发的突变菌株可用于其他植物和动物多胺交换器家族的功能分析。我们还假设,只要这些蛋白质可以在细菌细胞膜中表达,这种方法就可以用来描述许多其他类型的抗剂。大肠杆菌是一种对新型运输者进行表征的良好系统,因为有多种方法可用于诱变性原生运输者。

Introduction

参与代谢物贩运的蛋白质构成生理调节的基本水平,但绝大多数植物膜转运者尚未具有功能特征。已经实施了几种策略来描述新的运输蛋白。大肠杆菌和真核细胞(如酵母、异体卵母细胞、哺乳动物细胞、昆虫细胞和植物细胞)中的异体表达都被用来确定其迁移活性1。真核细胞是真核蛋白表达的宠儿,因为基本的细胞组成、信号转导途径、转录和翻译机与原生条件兼容。

酵母是植物中新型运输蛋白表征的重要模型。第一个植物运输蛋白,成功地表达在酵母(糖霉菌)是六氯苯(HUP1)从小球藻2。自那时以来,许多植物运输蛋白一直使用酵母表达系统进行功能特征化。其中包括植物糖运输机(SUC1和SUC2 3、VfSUT1和VfSTP14)和辅助糖运输机(AUX1和PIN5)。利用酵母表达植物蛋白的缺点包括,由于酵母缺乏这种细胞器,而蛋白原体局部蛋白的活性受损,靶向错误以及由于膜蛋白7、8、9的过度表达而在酵母中形成错误折叠的聚集体和激活应激反应

异体表达在体细胞中的运输蛋白已被广泛用于运输器10的电生理表征。第一个植物运输蛋白,在Xenopus卵母细胞中使用异体表达为特征的是阿拉伯拟钾通道KAT110和阿拉伯拟南芥六角体运输机STP111。自那时以来,Xenopus卵母细胞已被应用于许多植物运输蛋白的特征,如血浆膜运输器12,真空蔗糖运输器SUT413和真空麻黄素运输机ALMT914。转移性卵母细胞用于运输测定的一个重要限制是细胞内代谢物的浓度不能纵1 。此外,需要专业知识来准备Xenopus卵母细胞,并且卵母细胞批次的变异性难以控制。

模型有机体大肠杆菌中的异构表达是新植物运输蛋白表征的理想系统。大肠杆菌的分子和生理特征是众所周知的。 分子工具和技术已经建立起来此外,不同的表达载体,非致病菌株和突变体可用17,18,19。此外,大肠杆菌具有高生长率,在实验室条件下很容易生长。许多蛋白质在大肠杆菌9中易于表达和纯化。当蛋白质不能直接在细胞系统中被测定时,将蛋白质重组成脂质体也是一次成功的创新,尽管对于纯化膜蛋白的表征来说也是一项成功的创新。利用该模型系统20、21,完成了植物线粒体运输蛋白的功能表征,包括大豆、玉米、水稻和阿拉伯兰多普西的磷酸盐输送剂等溶质运输剂,在阿拉伯拟南芥中实现了二甲苯甲酸酯-三甲苯酯载体的功能特征。然而,在重组实验中发现番茄蛋白SICAT9的重组蛋白不起作用,而CAT运输体家族的其他成员在Xenopus卵母细胞测定22中被发现无功能。因此,需要额外的分子工具来表征膜输送机。

在大肠杆菌23中发现了5个多胺运输系统。其中包括两名ABC运输商,负责调解精氨酸和普赖辛的摄入,一个放流剂/苯基交换器,一个尸体/赖氨酸交换器,一个精氨酸出口商和一个放流剂进口商。Putrescine交换器PotE最初使用囊泡测定,其中内向外囊泡由裂化细胞与法国印刷机和测量放射性标记putresin到囊泡的吸收,以换取卵泡24。囊泡测定也用于表征钙输送机,该因子在质子梯度25的响应下调节钙的传递。这些实验促使我们制定了一种对其他多胺交换器进行表征的策略。我们首先在PotECadB交换器中制造了大肠杆菌缺乏菌株。在这里,我们演示了植物多胺抗剂的功能表征,通过修饰后的大肠杆菌菌株中的异质表达、使用法国印刷机生成膜囊泡以及放射性标记检测。

Protocol

1. 生成具有 P1 转导的大肠杆菌双敲出突变剂 从大肠杆菌遗传存量中心(http://cgsc.biology.yale.edu)获得大肠杆菌单敲除突变菌株[PotE和+CadB]注:βPotE菌株耐卡那霉素26,βCadB菌株抗四环素27。 使用P1转导协议28构建PotE/CadB</e…

Representative Results

此协议中的主要步骤在图 1中进行了图中的图中概述。简单地说,所有聚胺交换器和表达AtBAT1中缺乏的大肠杆菌细胞被培养、离心、用缓冲液清洗,并使用法国印刷机进行细胞解压。莱西斯倾向于产生囊泡,主要是从内到外,并捕获细胞外的缓冲液。细胞碎片通过离心去除,第二个超离心步骤用于收集膜颗粒。膜颗粒在Tris-Maleate缓冲液pH…

Discussion

在本研究中,我们概述了一种对抗分子进行表征的方法,首先在大肠杆菌中表达蛋白质,然后生成膜囊泡,从而可以在无细胞系统中对异位表达的蛋白质进行测定。除了在大多数分子生物学实验室中发现的设备外,这一策略还要求使用法国压力机、超离心机和进入进行放射性同位素检测的设施。

该技术的一个基本要求是异质蛋白正确定位在大肠杆菌的血浆膜上?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这个项目的支持来自BGSU研究生院和BGSU赞助项目和研究办公室。

Materials

2-mercaptoethanol Sigma-Aldrich M6250
3H-putrescine PerkinElmer NET185001MC
3H-spermidine PerkinElmer NET522001MC
4-chloro-1-naphthol Sigma-Aldrich C8890
14C arginine Moravek Inc. MC137
Arginine Sigma-Aldrich A-5006
Anti-His (C-term)-HRP antibody ThermoFisher R931-25 Detects the C-terminal polyhistidine (6xHis) tag, requires the free carboxyl
group for detection
Arabinose Sigma-Aldrich A3256
BCA protein assay kit ThermoFisher 23227 Pierce BCA protein asay kit.
Bromophenol blue Bio-Rad 161-0404
Carboxypeptidase B Sigma-Aldrich C9584-1mg
Centrifuge Sorvall SS-34 fixed angle rotor and GA-6 fixed angle rotor
Dounce tissue grinder LabGenome 7777-7 Corning 7777-7 pyrex homogenizer with pour spout.
Ecoscint-H National Diagnostics LS275 scintillation cocktail
EDTA Sigma-Aldrich
Filtration manifold Hoefer FH225V
French Pressure Cell Glen Mills FA-080A120
GABA Sigma-Aldrich A2129
Glutamate Sigma-Aldrich G6904
Glycerol
GraphPad Prism software http://www.graphpad.com/prism/Prism.htm
Hydrogen peroxide KROGER
Potassium Chloride J.T. Baker 3040-01
Liquid scintillation counter Beckman LS-6500
Maleate Sigma-Aldrich M0375
Nanodrop ThermoFisher
Nitrocellulose membrane filters Merck Millipore hawp02500 0.45 µM
PCR clean up kit Genscript QuickClean II
Potassium Phosphate dibasic ThermoFisher P290-500
putrescine fluka 32810
Potassium Phosphate monobasic J.T.Baker 4008
Spermidine Sigma-aldrich S2501
Strains :E. coli ΔpotE740(del)::kan, ΔcadB2231::Tn10 This manuscript Available upon request. Strain is deficient in the PotE and CadB polyamine exchangers.
Tris-base Research Products T60040-1000
Ultracentrifuge Sorvall MTX 150 46960 Thermo Fisher S150-AT fixed angle rotor
Ultracentrifuge tubes ThermoFisher 45237 Centrifuge tubes for S150-AT rotor
Vector: pBAD-DEST49 ThermoFisher Gateway expression vector for E. coli

References

  1. Haferkamp, I., Linka, N. Functional expression and characterisation of membrane transport proteins. Plant Biology (Stuttgart). 14 (5), 675-690 (2012).
  2. Sauer, N., Caspari, T., Klebl, F., Tanner, W. Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe. Proceedings of the National Academy of Sciences of the United States of America. 87 (20), 7949-7952 (1990).
  3. Sauer, N., Stolz, J. SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. The Plant Journal. 6 (1), 67-77 (1994).
  4. Weber, H., Borisjuk, L., Heim, U., Sauer, N., Wobus, U. A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. Plant Cell. 9 (6), 895-908 (1997).
  5. Huang, J. G., et al. GhDREB1 enhances abiotic stress tolerance, delays GA-mediated development and represses cytokinin signalling in transgenic Arabidopsis. Plant, Cell & Environment. 32 (8), 1132-1145 (2009).
  6. Bassham, D. C., Raikhel, N. V. Plant cells are not just green yeast. Plant Physiology. 122 (4), 999-1001 (2000).
  7. Garcia-Mata, R., Bebok, Z., Sorscher, E. J., Sztul, E. S. Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. Journal of Cell Biology. 146 (6), 1239-1254 (1999).
  8. Liu, J., Sitaram, A., Burd, C. G. Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase. Traffic. 8 (10), 1375-1384 (2007).
  9. Drew, D., et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nature Protocols. 3 (5), 784-798 (2008).
  10. Schachtman, D. P., Schroeder, J. I., Lucas, W. J., Anderson, J. A., Gaber, R. F. Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science. 258 (5088), 1654-1658 (1992).
  11. Boorer, K. J., Forde, B. G., Leigh, R. A., Miller, A. J. Functional expression of a plant plasma membrane transporter in Xenopus oocytes. FEBS Letters. 302 (2), 166-168 (1992).
  12. Miller, A. J., Zhou, J. J. Xenopus oocytes as an expression system for plant transporters. Biochimica et Biophysica Acta. 1465 (1-2), 343-358 (2000).
  13. Reinders, A., Sivitz, A. B., Starker, C. G., Gantt, J. S., Ward, J. M. Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus. Plant Molecular Biology. 68 (3), 289-299 (2008).
  14. Kovermann, P., et al. The Arabidopsis vacuolar malate channel is a member of the ALMT family. The Plant Journal. 52 (6), 1169-1180 (2007).
  15. Blattner, F. R., et al. The complete genome sequence of Escherichia coli K-12. Science. 277 (5331), 1453-1462 (1997).
  16. Terpe, K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology. 72 (2), 211-222 (2006).
  17. Miroux, B., Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology. 260 (3), 289-298 (1996).
  18. Wagner, S., et al. Consequences of membrane protein overexpression in Escherichia coli. Molecular & Cellular Proteomics. 6 (9), 1527-1550 (2007).
  19. Bernaudat, F., et al. Heterologous expression of membrane proteins: choosing the appropriate host. PLoS One. 6 (12), e29191 (2011).
  20. Takabatake, R., et al. Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean, maize, rice, and Arabidopis. Plant Molecular Biology. 40 (3), 479-486 (1999).
  21. Picault, N., Palmieri, L., Pisano, I., Hodges, M., Palmieri, F. Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria. Bacterial expression, reconstitution, functional characterization, and tissue distribution. Journal of Biological Chemistry. 277 (27), 24204-24211 (2002).
  22. Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. A., Sweetlove, L. J. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition. The Plant Journal. 81 (5), (2015).
  23. Kashiwagi, K., Igarashi, K. Identification and assays of polyamine transport systems in Escherichia coli and Saccharomyces cerevisiae. Methods in Molecular Biology. 720, 295-308 (2011).
  24. Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., Igarashi, K. Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 89 (10), 4529-4533 (1992).
  25. Tsuchiya, T., Rosen, B. P. Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli. Journal of Biological Chemistry. 251 (4), 962-967 (1976).
  26. Baba, T., et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology. 2, (2006).
  27. Nichols, B. P., Shafiq, O., Meiners, V. Sequence analysis of Tn10 insertion sites in a collection of Escherichia coli strains used for genetic mapping and strain construction. Journal of Bacteriology. 180 (23), 6408-6411 (1998).
  28. . P1vir phage transduction Available from: https://openwetware.org/wiki/Sauer:P1vir_phage_transduction (2011)
  29. . pBAD-DEST49 Gateway Destination Vector Available from: https://www.thermofisher.com/order/catalog/product/12283016 (2010)
  30. Green, M. R., Sambrook, J. . Molecular Cloning: a laboratory Manual. , (2012).
  31. Smith, P. K., et al. Measurement of protein using biochinic acid. Analytical Biochemistry. 150, 76-85 (1985).
  32. Wright, J. K., Overath, P. Purification of the lactose: H+ carrier of Escherichia coli and characterization of galactoside binding and transport. European Journal of Biochemistry. 138 (3), 497-508 (1984).
  33. . GaphPad Software Available from: https://www.graphpad.com/data-analysis-resource-center/ (2019)
  34. Kirchberger, S., et al. Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays. Journal of Biological Chemistry. 282 (31), 22481-22491 (2007).
  35. Deniaud, A., et al. Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy. Biochimica et Biophysica Acta. 1808 (8), 2059-2066 (2011).
  36. Sze, H. H+-Translocating ATPases: Advances Using Membrane Vesicles. Annual Review of Plant Physiology. 36, 175-208 (1985).
  37. Bush, D. R. Proton-coupled sugar and amino acid transporters in plants. Annual Review of Plant Physiology Plant Molecular Biology. 44, 513-542 (1993).
  38. Futai, M. Orientation of membrane vesicle from Escherichea coli prepared by different procedures. Journal of Membrane Biology. 115, 15-28 (1974).
  39. Seckler, R., Wright, J. K. Sideness of native membrane vesicles of Escherichea coli and orientation of the reconstituted lactose: H+ carrier. European Journal of Biochemistry. 142 (2), 269-279 (1984).
  40. LaVallie, E. R., Lu, Z., Diblasio-Smith, E. A., Collins-Racie, L. A., McCoy, J. M. Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods in Enzymology. 326, 322-340 (2000).
  41. Goodman, D. B., Church, G. M., Kosuri, S. Causes and effects of N-terminal codon bias in bacterial genes. Science. 342 (6157), 475-479 (2013).
  42. Wacker, M., et al. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science. 298 (5599), 1790-1793 (2002).

Play Video

Cite This Article
Ariyaratne, M., Ge, L., Morris, P. F. Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles. J. Vis. Exp. (154), e60009, doi:10.3791/60009 (2019).

View Video