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 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.

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% sucrose. Incubate at 30 °C O/N, rotating.
3. Dilute O/N culture so that the optical density at 600 nm measures 0.3 (OD₆₀₀ = 0.3) in 33 ml of selective media with 2% sucrose. Grow at 30 °C, shaking (Δ150 rpm).
4. When the culture has reached OD₆₀₀ = 0.8-1.5, induce expression of the desired protein by adding 17 ml of 3x YEP with 6% galactose (Recipe in Table 1), for a final concentration of 1x YEP with 2% galactose. Total culture volume is now 50 ml. Incubate, shaking, at 30 °C for an additional 5-6 hr.
   Note: the culture volume can be varied. In step 1.3, dilute into two-thirds of the desired final volume. In step 1.4, add one-third the final volume of 3x YEP/6% galactose.
5. Measure the OD₆₀₀ of the induced culture and centrifuge Δ150-200 OD₆₀₀ units of cells for 5 min at 5,000 rpm at 4 °C.
6. Resuspend cell pellet with 1 ml ice-cold 1x PBS with 1x protease inhibitor cocktail and transfer to a 2 ml screw cap tube.
7. Centrifuge cells for 1 min at 15,000 rpm at 4 °C. Decant supernatant.
8. Snap freeze cell pellet in liquid nitrogen, followed by immediate cell lysis or storage at -80 °C until further use.

2. Homogenization of Yeast Cells and Extraction of Proteins

1. To the frozen cell pellet from the previous step, add 200 µl of acid-washed glass beads and 500 µl of ice-cold lysis buffer (Recipe in Table 1 or use cell lysis buffer of choice).
2. Briefly pipette up and down. It is not required to fully resuspend the cell pellet. Keep tubes on ice at all times.
   Note: The end of the pipette tip may be cut off before pipetting up and down.
3. At 4 °C, place the tube(s) with cells into the bead mill, balance, lock, and run the machine as per manufacturer's instructions.
4. Run the bead beater containing the tube(s) for 20 sec at 5.5 m/sec, then place on slushy ice for 1 min. Repeat 6x in total.
5. Clear the extracted proteins by centrifugation for 15 min at 15,000 rpm at 4 °C. OPTIONAL: remove small particulates by centrifugation through a SpinX filter.
6. Prepare a sample of the whole cell extract (WCE) to confirm presence of the protein of interest by Western Blot:
   1. Add WCE corresponding with 2 OD₆₀₀ units of cells (e.g. 5 µl of cleared extract if 200 OD₆₀₀ units of cells were harvested) to 800 µl 20% trichloroacetic acid (TCA). Vortex to mix.
   2. Centrifuge for 2.5 min at 15,000 rpm at 4 °C. Decant the supernatant, but be careful to retain the pellet.
   3. Add 800 µl of 2% TCA to the pellet and vortex the tube, followed by centrifugation for 2.5 min at 15,000 rpm at 4 °C. Decant the supernatant, but be careful to retain the pellet.
   4. Add 100 µl of TCA Sample Buffer (Recipe in Table 1), vortex to dissolve pellet. Note: If the sample buffer becomes acidic (turns yellow) after addition to the pellet, add aliquots of Δ10 µl Tris base [1M] until the sample is blue again.
   5. Incubate in a 100 °C heat block for 2-5 min.
   6. Vortex again to fully dissolve if remnants of pellet are still present. Pellets prepared by this method are notoriously difficult to fully dissolve. It may take Δ10 min of vortexing to completely dissolve pellets.
   7. Store sample at -80 °C until further use.
7. Snap freeze aliquots of remaining cleared WCE in liquid nitrogen and store at -80 °C until further use.

3. Batch Purification of Proteins from Yeast Cell Homogenates

Note: This purification method was optimized for purification of 6xHIS-tagged proteins on Co²⁺ metal affinity resin.

1. Resin Equilibration
   1. For a sample of cleared WCE prepared from Δ20-40 OD₆₀₀ units of cells, add 50-100 µl of affinity resin to a microcentrifuge tube. Proteins extracted from cells that did not express the desired protein or a nonspecific resin, such as amylose, may be used as controls for nonspecific binding.
   2. Wash resin 5x with 1 ml of wash buffer: invert top-over-bottom until resin is resuspended, and then spin for 1 min at 5,000 rpm at 4 °C. Aspirate the supernatant.
      Note: if performing extraction and purification on the same day, resin equilibration can be performed prior to extraction.
2. Protein Binding for Affinity Purification
   1. Add 100-200 µl of cleared lysate (Δ20-40 OD₆₀₀ units) to 50-100 µl washed beads, and bring the total volume to 1 ml with lysis buffer.
   2. Incubate with nutation or rocking at 4 °C for 2-5 hr.
   3. Spin for 1 min at 5,000 rpm at 4 °C.
   4. If desired, save a sample of the remaining supernatant for subsequent analysis of unbound proteins. TCA precipitate as detailed above for the WCE (step 2.6).
   5. Wash resin with bound proteins 5x with 1 ml of wash buffer, followed by a spin for 1 min at 5,000 rpm at 4 °C. Keep samples cold during washes.
3. Elution of Bound Proteins
   1. Add 150 µl elution buffer to resin, nutate at 4 °C for 5 min, spin for 1 min at 5,000 rpm at 4 °C and save the supernatant in a new tube. OPTIONAL: Repeat 2x and pool elutions.
   2. Prepare eluted sample for Western blot: To 25 µl of eluted proteins, add 25 µl 2x lithium dodecyl sulfate (LDS) sample buffer with 2 µl β-mercaptoethanol (BME) and incubate in a 100 °C heat block for 2 min.
      Note: Standard 2X Laemmli sample buffer may also be used.
   3. Snap freeze excess eluted protein in liquid nitrogen.
      OPTIONAL: strip remaining proteins from resin with an equal volume of 2x LDS sample buffer at 65 °C for 5 min, remove the LDS-eluted proteins to a new tube, then add 2 µl BME to the eluted proteins, not to the resin. This order is to prevent removal of any ions or antibodies that are conjugated to the beads.
   4. Store samples at -80 °C until further use.
4. Western Blot and probe with appropriate antibodies to visualize proteins.
   1. Load 10-20 µl (Δ1-3 OD₆₀₀ units) of each sample and 3-10 µl of a protein ladder in an SDS-PAGE gel of choice. Gels routinely used for this protocol include 4-12% Bis-Tris and 8% Tris-Glycine.
   2. Run gel at 200 V for 50 min.
   3. Transfer proteins from gel to a PVDF membrane by semi-dry transfer at 19 V for 20-30 min (recipe in Table 1).
   4. Block membrane in 4% milk/1x Tris buffered saline-TWEEN (TBST) for 1 hr at RT or O/N at 4 °C (recipe in Table 1).
   5. Incubate membrane with primary antibody to the epitope-tagged protein of interest in 4% milk/1x TBST for 1-3 hr at RT or O/N at 4 °C.
   6. Wash membrane 3x for 5 min each with 1x TBST.
   7. Incubate membrane with appropriate secondary horseradish peroxidase (HRP) -conjugated antibody for 1-3 hr at RT.
   8. Wash membrane 3x for 15 min each in a large volume of 1x TBST.
   9. Cover membrane with ECL substrate and wrap in saran wrap.
   10. Expose membrane to film and develop to visualize proteins.

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 qualit...

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

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)