Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Summary

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Abstract

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

Introduction

The awesome power of yeast genetics is legendary, but the preparation and analysis of native proteins from budding yeast, Saccharomyces cerevisiae, is often fraught with problems. The latter is due to the considerable mechanical strength and elasticity of the yeast cell wall¹. Different means have been described for the enzymatic, chemical, mechanical, and pressure-based disruption of yeast cells to obtain whole-cell protein extract ^2-6. These techniques vary widely in their efficacy to yield cell-representative, native protein extracts that can be used for subsequent analyses or purification steps. For example, the yeast cell wall can b....

Protocol

Purification of 6xHIS-tagged proteins expressed in budding yeast cells under native conditions

1. Growth of Yeast Cells and Induction of Protein Expression

(Modified from ²)

OPTIONAL: Use logarithmically growing yeast cultures expressing protein of interest instead of the galactose-induced cultures described below.

1. Transform cells of a Gal⁺ yeast strain with a plasmid encoding a galactose-inducible 6xHIS-tagged protein of choice. For example, see reagents list.
2. Inoculate transformants in 5 ml of appropriate selective media (e.g. SD-uracil) containing 2% suc....

Results

Our representative results reveal that the described bead-beating and protein extraction protocol is useful for the reproducible preparation of proteins for a variety of downstream applications (summarized in Table 2). SDS-PAGE followed by Coomassie staining of WCEs show that a wide range of proteins (~12 to >250 kDa) can be reproducibly extracted from yeast cells (Figure 1A). Discrete bands over a range of molecular weights are indicative of high quality protein extracts. The quality.......

Discussion

This protocol focuses on the preparation of intact, native, and post-translationally modified proteins from budding yeast cells for down-stream applications. Before attempting this protocol, it is critical to determine if the protein of interest can be readily detected in protein extracts prepared under denaturing conditions¹². If polyclonal antibodies are not available it may be advantageous to epitope tag the protein of interest so that the fusion protein can be detected on Western blots. In the present prot.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Omni Bead Ruptor 24	Omni International	19-010
Yeast ORF strain in BG1805	ThermoScientific	YSC3869	GAL+ yeast strain that can be used for induction and extraction
pYES2.1 TOPO vector	Life Technologies	K4150-01	GAL+ yeast strain that can be used for induction and extraction; contains a V5/6xHIS tag
Halt Protease Inhibitor Single-Use Cocktail (100x)	Thermo Scientific	1860932
2.0 ml Skirted Tube with Tethered Screw Cap	BioExpress	C-3369-3	Make sure that the tube properly fits in the bead ruptor
Glass beads, acid-washed, 425-600 µm (30-40 US sieve)	Sigma-Aldrich	G8772
Corning Costar Spin-X centrifuge tube filters	Sigma-Aldrich	CLS8161	Use for additional filtering of clarified lysate
TALON Metal Affinity Resin	Clontech	635502	For the purification of 6xHIS tagged proteins
NuPAGE LDS Sample Buffer (4x)	Life Technologies	NP0007	To prepare and/or elute samples prior to SDS-PAGE and Western Blotting
NuPAGE 4-12% Bis-Tris Gel	Life Technologies	NP0321BOX	Precast gels often used for SDS-PAGE prior to Western Blotting
Simply Blue	Life Technologies	#LC6060	Protein gel stain
Mammalian Lysis Buffer	Promega	G9381	Alternative commercial lysis buffer
Anti-V5 agarose	Sigma	A7345	Method of immunoprecipitation
ECL	Millipore	WBKL S0 050
PVDF membrane	Millipore	IPVH00010
BIS-TRIS gels	Life Technologies	NP0321BOX
anti-myc antibody	Covance	mms-150R
secondary antibody	Abcam	ab97040	Goat pAb to mouse IgG (HRP)