Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

Mengxiao Ma; Shreya Goyal; Christopher  G. Burd; Richard  J. Chi

doi:10.3791/60413

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

JoVE Journal
Genetics

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JoVE Journal Genetics

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

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10:28 min

December 6, 2019

DOI: 10.3791/60413-v

Mengxiao Ma¹, Shreya Goyal², Christopher G. Burd¹, Richard J. Chi²

¹Department of Cell Biology,Yale School of Medicine, ²Department of Biological Sciences,University of North Carolina at Charlotte

Overview

This article presents a workflow for the expression, purification, and liposome binding of SNX-BAR heterodimers in yeast. The methods described facilitate the study of these membrane remodeling proteins, which are crucial in human disease.

Key Study Components

Area of Science

Neuroscience
Biochemistry
Cell Biology

Background

SNX-BARs are involved in membrane remodeling.
They play significant roles in various human diseases.
Understanding their properties can aid in therapeutic development.
Purification from yeast minimizes toxicity and insolubility issues.

Purpose of Study

To develop a reliable method for studying SNX-BAR proteins.
To determine the biophysical properties of SNX-BAR heterodimers.
To facilitate lipid binding and membrane remodeling studies.

Methods Used

Expression and purification of SNX-BAR proteins from yeast.
Characterization of lipid binding properties.
Use of correct extruder assembly to prevent liposome loss.
Visual aids for critical steps to assist beginners.

Main Results

Successful purification of native homo and hetero SNX-BAR dimers.
Methods applicable to all yeast SNX-BARs and other organisms.
Improved understanding of lipid specificities.
Visual observations enhance success rates for new researchers.

Conclusions

The developed workflow is effective for studying SNX-BAR proteins.
Insights gained can contribute to understanding membrane dynamics.
Methods can be adapted for broader applications in research.

Frequently Asked Questions

What are SNX-BAR proteins?

SNX-BAR proteins are membrane remodeling proteins that play key roles in various cellular processes and human diseases.

Why use yeast for purification?

Yeast allows for the acquisition of native protein dimers while minimizing toxicity and insolubility issues common in other expression systems.

What is the significance of lipid binding studies?

Understanding lipid binding is crucial for elucidating the mechanisms of membrane remodeling and the roles of SNX-BARs in cellular functions.

How can beginners improve their chances of success?

Visually observing critical steps in the workflow can greatly enhance understanding and execution of the methods.

Can these methods be applied to other organisms?

Yes, the methods can be adapted to produce recombinant SNX-BAR proteins from various organisms.

What is the role of the extruder assembly?

A correct extruder assembly is essential to prevent liposome loss during the extrusion process, ensuring successful experiments.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BARs are membrane remodeling proteins that play key roles in human disease. Using our methods, we can determine their precise biophysical properties to facilitate lipid binding and membrane remodeling. Purifying the SNB-BAR proteins from yeast cells allows the acquisition of native homo and hetero SNX-BAR dimers while minimizing the toxicity and insolubility frequently encountered with non-native expression systems.

Our methods can be used to understand the lipid specificities of all yeast SNX-BARs or to produce recombinant SNX-BAR proteins from any other organism. A correct extruder assembly is essential to preventing liposome loss during the extrusion process. For beginning students or those new to the field, visually observing the most critical steps will greatly improve your chances of success.

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