Name: budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications
Uploaded: 2013-10-30T22:35:00
Description: Watch this Scientific Journal Video about Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications at JoVE.com

We thank all members of the Kerscher lab for their support. We thank Mark Hochstrasser for yeast strain MHY3765. This work was supported by NSF grant 1051970 (to OK) and a Howard Hughes Medical Institute Undergraduate Travel grant and Monroe Scholars Program Grant (to ES).

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.
<ol>
	<li>Transform cells of a Gal+ yeast strain with a plasmid encoding a galactose-inducible 6xHIS-tagged protein of choice...

<ol>
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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...

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 be removed with lytic enzymes (e.g. zymolyase) and resulting spheroblasts can be disrupted by shearing, detergents, or osmotic lysis to release proteins. This approach has been successfully employed as the starting point for many subcellular fractionations but it requires lengthy incubations that are not compatible with the stability of some proteins7.
Proprietary yeast lysis reagents (such as detergents) for the chemical extraction of proteins of yeast cells are commercially available but the efficacy of these reagents in protein extraction and their effect on subsequent biochemical characterization of proteins is not always clear8. High pressure homogenizers, often referred to as French presses, effectively break yeast cells by first subjecting them to high pressure and then extruding them through a small opening in a pressure cell. This technique produces high quality extracts but the equipment is very expensive and may not be suitable for small quantities of cells or multiple samples9. Therefore, mechanical disruption of yeast cells in a bead mill is often the method of choice for native yeast protein preparations10. This technique involves mechanical disruption of the yeast cell wall with acid-washed glass beads, which can be conducted with a variety of shakers, vortexers or bead mills. Notably, this method can be used to simultaneously process multiple smaller samples (1 ml of cells or less). Many different beads or bead mill disruption matrices are now commercially available to disrupt almost any kind of cell type in 2 ml tubes. Considering the other techniques and equipment, a bead mill has the added advantage that the disruption of yeast cells occurs very fast, which helps to preserve post-translational modifications such as sumoylation, especially when the appropriate buffers with protease and/or proteasome inhibitors are utilized and the temperature of extracts is controlled.
This protocol focuses on the fast, effective, and reliable extraction of endogenous and over-expressed proteins under gentle conditions with the ultimate goal to preserve protein function, interactions, and post-translational modifications. Growth media, cell lysis buffer compositions, and bead mill settings are optimized to maintain protein interactions and post-translational modifications such as sumoylation and ubiquitylation.

<table border="0" cellpadding="0" cellspacing="0" width="1039">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Omni Bead Ruptor 24</td>
			<td style="width:124px;">Omni International</td>
			<td style="width:119px;">19-010</td>
			<td style="width:580px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast ORF strain in BG1805</td>
			<td>ThermoScientific</td>
			<td>YSC3869</td>
			<td>GAL+ yeast strain that can be used for induction and extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pYES2.1 TOPO vector</td>
			<td>Life Technologies</td>
			<td>K4150-01</td>
			<td>GAL+ yeast strain that can be used for induction and extraction; contains a V5/6xHIS tag</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Halt Protease Inhibitor Single-Use Cocktail (100x)</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">1860932</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.0 ml Skirted Tube with Tethered Screw Cap</td>
			<td>BioExpress</td>
			<td style="width:119px;">C-3369-3</td>
			<td>Make sure that the tube properly fits in the bead ruptor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid-washed, 425-600 &#181;m (30-40 US sieve)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Costar Spin-X centrifuge tube filters</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">CLS8161</td>
			<td>Use for additional filtering of clarified lysate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TALON Metal Affinity Resin</td>
			<td style="width:124px;">Clontech</td>
			<td>635502</td>
			<td>For the purification of 6xHIS tagged proteins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE LDS Sample Buffer (4x)</td>
			<td style="width:124px;">Life Technologies</td>
			<td>NP0007</td>
			<td>To prepare and/or elute samples prior to SDS-PAGE and Western Blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 4-12% Bis-Tris Gel</td>
			<td style="width:124px;">Life Technologies</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>Precast gels often used for SDS-PAGE prior to Western Blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Simply Blue</td>
			<td style="width:124px;">Life Technologies</td>
			<td style="width:119px;">#LC6060</td>
			<td>Protein gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mammalian Lysis Buffer</td>
			<td>Promega</td>
			<td style="width:119px;">G9381</td>
			<td>Alternative commercial lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-V5 agarose</td>
			<td>Sigma</td>
			<td style="width:119px;">A7345</td>
			<td>Method of immunoprecipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL</td>
			<td style="width:124px;">Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:124px;">Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BIS-TRIS gels</td>
			<td style="width:124px;">Life Technologies</td>
			<td>NP0321BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-myc antibody</td>
			<td style="width:124px;">Covance</td>
			<td style="width:119px;">mms-150R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibody</td>
			<td style="width:124px;">Abcam</td>
			<td style="width:119px;">ab97040</td>
			<td>Goat pAb to mouse IgG (HRP)</td>
		</tr>
	</tbody>
</table>

budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications

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&#160;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.

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 &#62;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 qualit...

Watch this Scientific Journal Video about Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications at JoVE.com

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

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.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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