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Biologia

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

Published: June 13, 2021
doi:
10.3791/62592
, , , , ,
1Sir John Walsh Research Institute, Faculty of Dentistry,University of Otago, 2Department of Microbiology, Faculty of Medicine,Chulalongkorn University, 3School of Biological Sciences,University of Auckland

Summary

本文提出了一个小规模的等离子膜隔离协议，用于描述在糖浆中过度表达的念珠菌ABC（ATP结合盒式）蛋白质Cdr1。 蛋白酶可切割的 C 端 mGFPHis 双标签，Cdr1 和标签之间有 16 残留链接器，旨在促进 Cdr1 的净化和洗涤剂筛选。

Abstract

ABC运输机的生化和生物物理特征的成功在很大程度上取决于异质表达系统的选择。在过去的二十年里，我们开发了一个酵母膜蛋白表达平台，用于研究许多重要的真菌膜蛋白。表达宿主 Saccharomyces cerevisiae AD+在七个主要的内源ABC转运器中被删除，它包含转录因子Pdr1-3与功能增益突变，使异质膜蛋白基因的构成过度表达稳定地整合为基因组 PDR5 位点的单拷贝。多功能质粒载体的创建和单步克隆策略的优化使异种ABC运输机能够快速准确地进行克隆、突变和表达。在这里，我们描述了一个新的蛋白酶可切割mGFPHis双标签（即单体酵母增强绿色荧光蛋白yEGFP3融合到六希丁亲和纯化标签）的发展和使用，旨在避免标签可能干扰感兴趣的蛋白质，并提高他的标签对镍亲和力树脂的结合效率。mGFPHis 与膜蛋白 ORF（开放读取框架）的融合，通过检测聚丙烯酰胺凝胶和检测保留 mGFPHis 标签的降解产品，可以轻松量化蛋白质。我们演示了此功能如何促进膜蛋白溶化的洗涤剂筛选。介绍了一个协议，高效，快速和可靠的隔离小规模等离子膜制剂的C终端标签 念珠菌 多药废水运输机Cdr1过度表达在 S.塞雷维西亚 AD+，提出了。这种小规模等离子膜隔离协议可在一个工作日内生成高质量的等离子膜。等离子膜制剂可用于确定Cdr1和Cdr1突变变异的酶活动。

Introduction

从原生脂质环境中提取整体膜蛋白会显著影响其结构和功能1、2、3、4。生物膜5的复杂脂质组成确保了至关重要的蛋白质-脂质相互作用可能发生6。脂质保持膜蛋白的结构完整性，从而使它们能够在膜室目标7，8正确运作。因此，膜蛋白纯化的关键第一步是从其原生环境中提取蛋白质，而不影响其结构和/或功能。

膜蛋白的结构存在许多障碍，其中大多数与膜蛋白的疏水性质有关，难以以X射线晶体学或低温电子显微镜（Cryo-EM）9、10、11、12所需的数量表达适当折叠和功能膜蛋白。膜蛋白表达系统有三种类型：同源性9、异质13、14、15和体外表达系统16、17。许多表达系统的表达水平往往很低，或成本过高，因此只有少数宿主成为生产膜蛋白的首选。它们包括细菌宿主，大肠杆菌，酵母S.塞雷维西亚和皮希亚面食，和更高的真核生物，如Sf9昆虫细胞或哺乳动物细胞系18。所有膜蛋白表达技术各有优缺点：然而，S.Cerevisiae也许是研究得最好的真核模型有机体，适合膜蛋白的生产。它具有高度多功能性，在基因工程、药物发现、合成生物学和真核膜蛋白14、19、20、21的表达方面具有很强的应用性。

在这项研究中，使用了一项获得专利的S.cerevisiae膜蛋白表达技术21，以S.Cerevisiae AD+14和AD+22为首选宿主（图1A），以过度表达和研究主要的C.藻类多药排泄泵Cdr1。S. cerevisiae菌株均为 AD1-8u–23的衍生物，具有ura3 （AD+） 或ura3及其1 （AD+） 基因，以消除任何通过URA3或HIS1基因组洛奇的不需要整合产生的假阳性尿素或组织丁原体转化剂。图1A中指出的7种主要多药排泄物泵23的删除，使得AD+对大多数异种生物非常敏感。功能增益突变转录因子Pdr1-3通过两个同源重组事件整合了基因组PDR5点的异质-ORF转化盒（图1A）后，导致Cdr1（图1A中的红色八角形）等异质膜蛋白的构成过度表达。C-终端mGFPHis标记蛋白的适当等离子膜定位可以通过共聚焦显微镜（图1A）确认，他的标签可用于标记蛋白质的镍亲和纯化。然而，将一些真菌ABC运输机（例如，坎迪达·克鲁塞ABC1）克隆成pABC3衍生质粒是不可能的，因为它们由于细胞毒性而无法在大肠杆菌中传播。这促使膜蛋白14、24的单步克隆的发展，这些蛋白在N-或C终点站被标记为具有各种亲和力、表位或记者标签，直接进入S.Cerevisiae AD+（图1C）。塞雷维西亚AD+菌株过度表达各种CDR1突变体也可以有效地创建这种方式，使用多达5个单独的PCR片段重叠25个基点（图1C）。利用此协议，许多感兴趣的ORF可以在很短的时间内以低成本和高效率进行克隆、表达和定性。每增加一个 PCR 片段，转化效率只会降低 ±2 倍。 

如果需要，表达水平也可以很容易地通过入门设计操纵，以可预测地将表达水平调低到通常高，构成表达水平25的0.1%-50%之间。优化的，多功能的， pABC314衍生克隆载体， pABC3 – XLmGFPHis26 （图 1B）包含一个 HRV – 3C 蛋白酶位点（ X ：列夫夫克|GP），一种蛋白酶，在4°C下表现优于常用的烟草蚀刻病毒（TEV）蛋白酶27。L是一种五氨基酸（GSGGS）链接器，mGFP是一种单体突变体（A206K）28、29版酵母增强绿荧光蛋白变异yEGFP33 30，而他的是一个三氨基酸连结剂（GGS），其次是六异丁（HHHHH）镍亲和蛋白纯化标签。

这种表达技术已成功地应用于药物发现和膜蛋白的研究。真菌偶佐尔药物靶点的第一个结构，S.塞雷维西亚Erg1131，是利用这项技术解决的。它还使C.藻类Cdr132，33，34的详细特征和创建一个赛斯坦缺乏Cdr1分子35适合赛尔坦交叉链接研究，以验证任何未来的高分辨率结构。许多其他ABC运输机从主要的人类真菌病原体（即， C. 阿尔比肯斯，坎迪达格拉布拉塔，坎迪达奥里斯，坎迪达克鲁塞，坎迪达使用，隐球菌新福尔曼斯，阿斯珀吉卢斯富米加图斯，青霉素马内菲，和富萨里姆索拉尼物种复合体）也已使用这个表达平台24，36，37，38，39详细研究。这使得在高通量屏幕上使用的S.cerevisiae菌株的面板能够过度表达的污水泵，从而发现新型荧光浮流泵基材尼罗河红色40和特异性41和广谱14、33、42、43、44 efflux泵抑制剂。该系统的使用也使发现木糖线作为同类广谱真菌多药排泄泵抑制剂42。

膜蛋白的完全溶化和创建无内源脂质的同质膜蛋白-鼠类制剂，需要高洗涤剂浓度45。但不幸的是，这也经常灭活膜蛋白5，8，45，46。洗涤剂单体的特性和溶液中的聚合受疏水尾部的物理特性、烷基链的长度和分支、芳香核或氟烷基侧链的存在或聚氧乙烯单元的数量的影响。因此，洗涤剂筛选是确定最适合膜蛋白溶解和纯化的洗涤剂的重要第一步。

C.藻类是人体免疫功能低下的主要真菌病原体，可引起严重、危及生命的侵入性感染47种，可对偶祖抗真菌药物产生抗药性48、49。C.藻类耐多药性的主要机制之一是Cdr150的过度表达，这是位于等离子膜中的ABCG亚家庭II型ATP绑定盒式（ABC）运输机51。全尺寸真菌 ABCG 运输机（由两个核苷酸结合域 [NBD] 和两个跨膜域 [TMD] 组成） 更通常被称为全热带耐药性 （PDR） 运输机，其特点是其独特的倒置域拓扑 [NBD-TMD]2.PDR运输机只在植物52，53和真菌54发现。尽管PDR运输机的重要性，没有结构，虽然结构为人类半尺寸ABG运输机最近已经解决，这有助于创造Cdr133的第一个试探性模型。然而，我们最近的实验证据表明，这种模型存在缺陷，可能是因为真菌PDR运输机具有特有的不对称性NBD，很可能形成一种独特的运输机制。因此，Cdr1 的高分辨率结构既需要合理设计有助于克服以埃夫卢斯为媒介的耐药性的新颖的 efflux 泵抑制剂，又需要深入了解这一重要的 ABC 运输机系列的行动机制。

本研究的目的是为转基因 的S.Cerevisiae 表达宿主的Cdr1表达、溶解和纯化制定可靠的协议，最终目的是为Cdr1获得高分辨率结构。作为此过程的一部分，一个蛋白酶可切割的 mGFPHis 双标签 （图 1B）设计与 16 残渣链接器分离标签从 Cdr1 的 C 总站， 这改善了附加的 6x 他的亲和力标签与镍亲和树脂的结合， 并使监测 Cdr1 表达水平在活细胞和整个净化过程中.还开发了含有约 10%C.藻类 Cdr1（根据SDS-PAGE之后库马西染色估计）的小规模酵母血浆膜蛋白制剂的可重复协议，可用于Cdr1的生化表征。

Protocol

1. 准备新鲜或冷冻的转化库存，使AD+和AD+细胞具有能力 接种 25 mL 的 2 倍 Ypcd [即 2 倍 Ypd; 2% （w/v） 酵母提取物， 2% （w/v） 丙酮， 4% （w/v） 德克斯特罗斯）， 0.079% （w/v） CSM （完全补充混合物）]35 介质与单个酵母菌落和孵育过夜 （o/n） 16 小时在 30 °C 与摇晃在 200 转每分钟 （rpm）.。 接种 225 mL 的 2x YPCD 介质与 25 mL o/n 培养，并检查细胞光学密度在 600 nm （OD…

Representative Results

使用 pYES2（图 2B）实现了S. cerevisiae AD+的高频率转换 （+4 x10 4变压剂/μg）。不出所料，无DNA（即仅限ddH2O）控制没有给出变压剂，线性CDR1-mGFPHis转换盒式磁带（图1A）的1微克给+50变压器（图2C）与优化的AD+转换协议。CDR1-mGFPHis变压剂还测试了它们在不可发酵碳源上生长的能力，以?…

Discussion

尽管膜蛋白结构分析最近取得了进展，但目前尚无Cdr1或任何其他PDR运输机的3D结构。因此，了解Cdr1结构及其生化特征非常重要，因为这不仅能深入了解新药的合理设计，克服以流体为媒介的耐药性，而且能深入了解ABC蛋白重要亚系的功能机制。

膜蛋白结构特征的主要要求之一是正确折叠和完整膜蛋白的表达，其数量为X射线结晶或低温-EM所需。选择表达系统时的重要标准是易…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感激地感谢新西兰马斯登基金（Grant UOO1305）的资助，以及来自泰国曼谷朱拉隆功大学医学院的整笔赠款（ M. Niimi）。他们要感谢奥塔哥大学向马达尼大学提供博士学位奖学金。作者还感谢斯特凡·劳恩瑟教授及其同事阿米尔·阿佩尔鲍姆博士和迪瓦纳亚加巴拉蒂·维纳亚加姆博士在德国多特蒙德马克斯·普朗克分子生理学研究所（MPIMP）对G·马达尼进行为期6个月的访问期间给予的支持和监督。作者还感谢德国学术交流局（DAAD）向G.马达尼提供了一项研究补助金（57381332），以访问MPIMP。

Materials

2-(N-Morpholino)ethane-sulphonic acid (MES) Sigma-Aldrich M3671
2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base; ultra-pure) Merck 77-86-1
2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside Anatrace NG310S LMNG
2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside Anatrace NG311S OGNG (MNG-OG)
2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside Anatrace NG322S DMNG
4-Trans-(4-trans-propylcyclohexyl)-cyclohexyl α-D-maltopyranoside Glycon Biochemicals GmbH D99019-C PCC-α-M
40% Acrylamide/Bis-acrylamide (37.5:1) Bio-Rad 1610148
Acetic acid (glacial) Merck 64-19-7
Agar Formedium  009002-18-0
Ammonium molybdate Sigma-Aldrich 13106-76-8
Ammonium persulphate (APS) Bio-Rad 1610700
ATP disodium salt sigma-Aldrich A-6419
Bromophenol blue SERVA Electrophoresis GmbH 34725-61-6
CHAPS Anatrace C316S
CHAPSO Anatrace C317S
CSM Formedium DCS0019
CSM minus uracil Formedium DCS0161
Cyclohexyl-1-butyl-β-D-maltopyranoside Anatrace C324S CYMAL-4
Cyclohexyl-1-heptyl-β-D-maltopyranoside Anatrace C327S CYMAL-7
Cyclohexyl-methyl-β-D-maltopyranoside Anatrace C321S CYMAL-1
Digitonin Sigma-Aldrich 11024-24-1
Dithiothreitol (DTT) Roche Diagnostics 10197785103
DMSO Merck 67-68-5
Ethanol Merck 459836
Ethylenediaminetetraacetic acid disodium salt (EDTA; Titriplex III) Merck 6381-92-6
ExoSAP-IT PCR Product Cleanup Reagent Applied Biosystems 78205 A blend of exonuclease and phosphatase
Glucose Formedium 50-99-7
Glycerol Merck 56-81-5
Glycine Merck G8898
HEPES Formedium 7365-45-9
Hydrochloric acid Merck 1003172510
KOD Fx Neo TOYOBO Co KFX-201 Use for reliable colony PCR
lithium acetate (LiAc) Sigma-Aldrich 546-89-4
Magnesium chloride hexa-hydrate sigma-Aldrich M2393
MES Formedium 145224-94-8
n-Decanoyl-N-hydroxyethyl-glucamide Anatrace H110S HEGA-10
n-Decanoyl-N-methyl-glucamide Anatrace M320S MEGA-10
n-Decyl-phosphocholine Anatrace F304S Fos-choline-10
n-Decyl-β-D-maltopyranoside Anatrace D322S DM
n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate Anatrace AZ312S Anzergent 3-12
n-Dodecyl-N,N-dimethylamine-N-oxide Anatrace D360S LDAO
n-Dodecyl-α-D-maltopyranoside Anatrace D310HA α-DDM
n-Dodecyl-β-D-maltopyranoside Anatrace D310S β-DDM
n-Nonyl-β-D-glucopyranoside Anatrace N324S NG
n-Nonyl-β-D-maltopyranoside Anatrace N330S NM
n-Octadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate Anatrace AZ318S Anzergent 3-18
n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate Anatrace AZ308S Anzergent 3-8
n-Octyl-phosphocholine Anatrace F300S Fos-choline-8
n-Octyl-β-D-glucopyranoside Anatrace O311S OG
n-Tetradecyl-phosphocholine Anatrace F312S Fos-choline-14
n-Tetradecyl-β-D-maltopyranoside Anatrace T315S TDM
n-Tridecyl-phosphocholine Anatrace F310S Fos-choline-13
n-Tridecyl-β-D-maltopyranoside Anatrace T323S –
n-Undecyl-β-D-maltopyranoside Anatrace U300S UM (UDM)
N,N,N’,N’-tetramethyl-ethylenediamine (TEMED) Sigma-Aldrich T9281
Octylphenoxypolyethoxyethanol Sigma-Aldrich 9002-93-1 TRITON X-100
Oligomycin Sigma-Aldrich 75351
Peptone Formedium 3049-73-7
phenylmethylsulfonyl fluoride (PMSF) Roche Diagnostics 329-98-6
Phusion Hot Start Flex DNA Polymerase New England Biolabs M0535S High-fidelity DNA polymerase
polyethylene glycol (PEG 3350) Sigma-Aldrich 25322-68-3
polyoxyethylenesorbitan monooleate Sigma-Aldrich 9005-65-6 TWEEN 80
Potassium nitrate Sigma-Aldrich P8394
Protein Assay Kit Bio-Rad 5000122 RC DC Protein Assay Kit II
QC Colloidal Coomassie Stain Bio-Rad 1610803
Prism Ultra Protein Ladder (10-245 kDa) Abcam AB116028
Sodium azide Sigma-Aldrich 71289
Sodium dodecyl sulphate Sigma-Aldrich 151-21-3 SDS
Sodium L-ascorbate BioXtra Sigma-Aldrich 11140
Sucrose Monododecanoate Anatrace S350S DDS
Sulphuric acid Sigma-Aldrich 339741
Yeast extract Formedium 008013-01-2
Yeast nitrogen base without amino acids Formedium CYN0402
 Equipment (type)
Centrifuge  (Eppendorf 5804) Eppendorf
Centrifuge (Beckman Ultra) Beckman
Centrifuge (Sorvall RC6) Sorvall
FSEC apparatus (NGC Chromatography Medium Pressure system equipped with a fluorescence detector, an autosampler, a fractionator) Bio-Rad
Gel imaging (GelDoc EZ Imager) Bio-Rad
Microplate reader (Synergy 2 Multi-Detection) BioTek Instruments
PCR thermal cycler (C1000 Touch) Bio-Rad
Power supply (PowerPac) Bio-Rad
SDS PAGE (Mini-PROTEAN Tetra) Bio-Rad
Shaking incubator (Multitron) Infors HT, Bottmingen
Superose 6 Increase 10/300 GL GE Healthcare Life Sciences GE17-5172-01
UV/Visible spectrophotometer (Ultraspec 6300 pro) Amersham BioSciences UK Ltd
DOWNLOAD MATERIALS LIST

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Plasma MembraneCandida AlbicansCdr1-mGFPHisMembrane ProteinSmall-scale IsolationATPaseDetergentCell HarvestingPre-cultureCell LysisCentrifugationHomogenization Buffer

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Madani, G., Lamping, E., Lee, H. J., Niimi, M., Mitra, A. K., Cannon, R. D. Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis. J. Vis. Exp. (172), e62592, doi:10.3791/62592 (2021).
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