Small-Scale Plasma Membrane Preparation for the Analysis of <em>Candida albicans</em> Cdr1-mGFPHis

Golnoush Madani; Erwin Lamping; Hee Ji Lee; Masakazu Niimi; Alok K. Mitra; Richard D. Cannon

doi:10.3791/62592

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Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

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09:44 min

June 13, 2021

DOI: 10.3791/62592-v

Golnoush Madani*¹, Erwin Lamping*¹, Hee Ji Lee¹, Masakazu Niimi^1,2, Alok K. Mitra³, Richard D. Cannon¹

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

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Overview

This study presents a small-scale plasma membrane isolation protocol aimed at characterizing the ABC protein Cdr1 in the eukaryotic model organism Saccharomyces cerevisiae. Key advantages include the method's rapidity, economy, and reliability in assessing membrane protein expression and ATPase activities.

Key Study Components

Research Area

Cell biology
Membrane protein characterization
Eukaryotic model systems

Background

Importance of ATP-binding cassette (ABC) proteins
Challenges in membrane protein purification
Role of Saccharomyces cerevisiae as a model organism

Methods Used

Small-scale plasma membrane isolation protocol
Saccharomyces cerevisiae
Protease-cleavable C-terminal mGFPHis double tag

Main Results

Successful characterization of Cdr1 using the new protocol
Effective measurement of membrane protein expression levels
Assessment of ATPase activities and optimal detergent usage

Conclusions

The protocol enhances the ease of membrane protein studies
Provides a valuable tool for future research involving membrane proteins

Frequently Asked Questions

What is the significance of the ABC protein Cdr1?

Cdr1 plays a crucial role in drug resistance and membrane transport in fungi.

Why is Saccharomyces cerevisiae used as a model organism?

It is an established model for studying eukaryotic cell biology and genetics.

How does the mGFPHis tag facilitate protein purification?

The mGFPHis tag allows for easy detection and purification of target proteins via fluorescence.

What are the advantages of the small-scale protocol?

It is economical, fast, and reliable, making it user-friendly for researchers.

What factors are critical for optimizing protocol success?

Harvesting cells at an OD 600 of 2 is essential for minimizing contamination and optimizing ATPase activity.

What downstream applications does this method enable?

The protocol allows for the investigation of membrane protein functions and interactions in various biological contexts.

Are there limitations to this isolation protocol?

Care must be taken to optimize conditions for specific membrane proteins, as variations may affect yield and activity.

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。

小尺度等离子体膜分离协议和mGFPHis双标签，提供了一个经济，快速，可靠，简单的方法来描述膜蛋白在真核模型生物体，糖体切口。这项技术的最大优点是膜蛋白表达水平、ATP的活动和洗涤剂最适合其纯化的易用性和速度。这项技术非常方便用户使用。

然而，需要记住的一个重要点是将细胞收获为OD 600的2，这确保了低线粒体污染和尽可能高的ATPase活动。首先，在30摄氏度的10毫升YPD介质中预先培育一个酵母菌群，每分钟200次旋转7至8小时。使用 10 毫升预培养物为 40 毫升新鲜 YPD 介质接种疫苗。

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