Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

Summary

We describe a reliable method for the preparation of whole cell extracts from yeast or other cells using a cryogenic freezer mill that minimizes degradation and denaturation of proteins. The cell extracts are suitable for purification of functional protein complexes, proteomic analyses, co-immunoprecipitation studies and detection of labile protein modifications.

Abstract

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN₂) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 °C achieved with the use of LN₂ protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN₂ to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN₂ in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.

Introduction

Yeast is a popular model organism for protein studies, as it is a simple eukaryotic organism with an abundance of genetic and biochemical tools available for researchers¹. Because of their sturdy cell wall, one challenge that researchers face is in efficiently lysing the cells without damaging the cellular contents. Different methods are available for obtaining protein extracts through disruption of yeast cells which include enzymatic lysis (zymolyase)^2,3, chemical lysis⁴, physical lysis by freeze-thaw⁵, pressure-based (French press)^6,7, mechanical (glass beads, coffee grinder)^8,9, sonication-based¹⁰ and cryogenic^2,11. The efficiency of cell lysis and the protein yield can vary considerably depending on the technique employed, thus affecting the end result or suitability for the desired downstream application for the lysate. When studying proteins that are unstable, have fleeting posttranslational modifications, or are temperature sensitive, it is particularly important to use a method that will minimize sample loss or degradation during preparation.

Extract preparation technique	Details	Advantages	Disadvantages	Downstream analysis	Reference
French press: High-pressure homogenizer (aka Microfluidizer) with enzymatic pretreatment using Zymolyase	Zymolyase-20T, a Microfluidizer high-pressure homogenizer. The disruptor consists of an air-driven, high-pressure pump (ratio 1:250; required air pressure 0.6-l MPa) and a special disruption chamber with an additional back pressure unit. A minimum sample size of 20 mL is required for processing.	Final total disruption obtained using the combined protocol approached 100 % with 4 passes at a pressure of 95 MPa, as compared to only 32 % disruption with 4 passes at 95 MPa using only homogenization without the Zymolyase.	Not appropriate for small scale applications. The enzymes can get expensive for large scale preparations.	Protein purification	6
Bead beater: Zymolyase treated cells lysed with glass beads in a fastprep instrument	Roughly an equal volume of cold, dry, acid-washed 0.5 mm glass beads is added to a given volume of cell pellet in lysis buffer and the cells are disrupted by vigorous manual agitation.	It is particularly useful when making extracts from many different small yeast cultures for assaying purposes rather than for protein purification.	During the glass bead procedure, proteins are treated harshly causing extensive foaming leading to protein denaturation. The amount of cell breakage varies, while proteolysis as well as modification of the proteins may result from heating of the extract above 4°C during the mechanical breakage.	Mostly DNA & RNA analyses, but also protein analysis by denaturing gel electropheoresis, either with or without Western blotting.	8
Zymolyase treatment   followed by lysis using a combination of osmotic shock and Dounce homogenization	After enzymatic digestion of cell walls, spheroplasts are lysed with 15 to 20 strokes of a tight-fitting pestle (clearance 1 to 3 µm) in a Dounce homogenizer.	Advantageous to use protease-deficient strains such as BJ926 or EJ101. This is the gentlest way to break yeast cells and hence it is most suitable for preparing extracts that can carry out complex enzymatic functions (e.g., translation, transcription, DNA replication) and in which the integrity of macromolecular structures (e.g., ribosomes, splicesomes) has to be maintained. It is also useful for isolating intact nuclei that can be used for chromatin studies (Bloom and Carbon, 1982) or for nuclear protein extracts (Lue and Kornberg, 1987).	The major disadvantages of the spheroplast lysis procedure are that it is relatively tedious and expensive, especially for large-scale preparations (>10 liters), and the long incubation periods can lead to proteolysis or protein modification.  For chromatin preparations, they seem to be of varying or lower quality than those produced by the differential centrifugation (based on nucleosome ladder integrity).	Isolating intact nuclei for chromatin studies, extracts that can carry out complex enzymatic functions, extracts requiring the integrity of macromolecular structures, nuclear protein extracts.	2
Cell Disruption of flash frozen cells by grinding in Liquid Nitrogen using a mortar/pestle or a blender	Cells are frozen immediately in liquid nitrogen and then lysed by grinding manually in a mortar using a pestle, or using a Waring blender in the presence of liquid nitrogen.	The protocol is quick and easy. It can accommodate varying amounts of yeast cells including very large cultures. Its main advantage is that cells are taken immediately from the actively growing state into liquid nitrogen (−196°C), decreasing degradative enzyme activities such as proteases and nucleases as well as activities that modify proteins (e.g., phosphatases and kinases). It is particularly suited for making whole-cell extracts from a single yeast culture for large-scale protein purification.	A bit messy and potentially dangerous to the careless investigator. Small samples (i.e., 10- to 100-ml yeast cultures) are not easily processed because there is not enough mass of frozen cell clumps to fracture effectively in the blender. It is time-consuming to process individual samples and to clean the equipment between uses.	Whole-cell extracts from a single yeast culture for large-scale protein purification.	2
Autolysis, Bead mill	pH 5.0, 50 °C, 24 h, 200 rpm / Ø 0.5 mm, 5 × 3 min/3 min	Quick and efficient lysis, especially for small scale extract preparation	Heat generation leads to denaturation and degradation of macromolecules. Bead beating equipment required.	Small scale analyses.	10
Autolysis, Sonication	pH 5.0, 50 °C, 24 h, 200 rpm, 4 × 5 min/2 min, pulser 80%, power 80%	Sonication equipment is usually available in most institutions.	Heat generation leads to denaturation and degradation of macromolecules. Sonication equipment required. Slow lysis can take more than 24 hours.	Yeast cell wall preparations.
Boiling and freeze-thaw process	No specialized equipment needed other than a standard freezer and a heating block or hot water bath.	Efficient, reproducible, simple and inexpensive.	Heat generation leads to denaturation and degradation of macromolecules.	DNA analyses by PCR.	5

Table 1: Comparison of methods available for the preparation of yeast extracts.

Cryogrinding (aka cryogenic grinding/cryogenic milling) is commonly employed to retrieve nucleic acids, proteins or chemicals from temperature sensitive samples in a reliable manner for quantitative or qualitative analyses. It has been used successfully for multiple applications in diverse fields including biotechnology, toxicology, forensic science^12,13, environmental science, plant biology¹⁴ and food science. Isolation of intact biological macromolecules is usually critically dependent on the temperature. Extremely low temperatures ensure that the proteases and nucleases stay inactive, resulting in a reliable isolation of intact proteins, nucleic acids and other macromolecules for subsequent analyses. Indeed, a freezer mill typically maintains a sample temperature of -196 °C (the boiling point of LN₂), thus minimizing DNA/RNA or protein denaturation and degradation.

The freezer mill employs an electromagnetic grinding chamber that rapidly moves a solid metal bar or cylinder back and forth within a vial containing the sample to be pulverized between stainless steel end plugs. The instrument creates and rapidly reverses a magnetic field within the grinding chamber. As the magnetic field shifts back and forth, the magnet crushes the sample against the plugs thus achieving the 'cryogrinding' and the pulverization of the popcorn. The freezer mill replaces the mortar and pestle and allows the sequential processing of multiple samples (or up to 4 smaller samples simultaneously) with high reproducibility and avoids the user-to-user variability associated with manual grinding. Once the samples are processed, the cell extracts can be used for a variety of downstream applications.

Protocol

1. Preparation of Yeast Popcorn

1. Grow yeast cells in 0.5 L of YPD media to a density of 1 x 10⁷ cells/mL. Count cells using a Coulter Counter or any other means.
2. Centrifuge cells for 10 min at 2,400 g and 4 °C.
3. Wash each sample once with 500 mL of ice-cold deionized 18 mega Ohm Milli-Q water.
4. Resuspend pellets in 15 mL of ice-cold Lysis Buffer [20 mM HEPES-KOH pH 7.5, 110 mM KCl, 0.1% Tween, 10% glycerol, with freshly added reducing agent 10 mM β-mercaptoethanol, protease inhibitor cocktail, 10 μM proteasome inhibitor MG-132, 1 mM deacetylase inhibitor sodium butyrate and phosphatase inhibitors (1 mM sodium vanadate, 50 mM sodium fluoride, 50 mM sodium β-glycerophosphate)]. See the Table of Materials for additional details. Keep resuspended cells on ice until the samples are ready for the next step.
   NOTE: A wide variety of lysis buffers containing a number of non-ionic detergents can be used with the samples and specific inhibitors included in it based on the downstream application. Inclusion of protease and proteasomal inhibitors are crucial for preventing protein degradation, especially once the extracts are thawed.
5. Make snap frozen yeast popcorn by slowly adding the cell suspension one drop at a time using a pre-chilled serological pipette into one or more 50 mL centrifuge tubes kept on dry ice and filled with LN₂ until just below the rim. Top up liquid nitrogen in the tube frequently to compensate for its loss due to rapid evaporation. Work in a well-ventilated area to avoid the hazards associated with nitrogen asphyxiation.
   1. Instead of a 50 mL tube, use multiple 50 mL tubes tied together (or any container with a large mouth, such as a large plastic centrifuge bottle that can tolerate cryogenic temperatures) for pop-corn preparation (Figure 1A). The larger the container and its opening, the easier is the popcorn preparation as this prevents clumping of the popcorn to form large aggregates. A size range of 0.3-0.5 cm for the popcorn is ideal for achieving proper grinding/pulverization using the freezer mill (Figure 1B).
   2. Ensure that the LN₂ has evaporated completely from the tubes containing the popcorn prior to storing them at -80 °C. It is possible to stop after preparing the yeast popcorn as they are stable for several years when maintained at -80 °C.

Figure 1: Yeast popcorn preparation. (A) Yeast "popcorn" is made by the dropwise freezing of the cell suspension in LN₂. We use one to three 50 mL tubes held together with a rubber band and placed in an ice bucket filled with dry ice. The tubes are filled with LN₂ until just below their rims and are topped up with liquid nitrogen frequently to keep them nearly full until all the cell suspension had been made into popcorn (B) The size of the popcorn is an important determinant of optimal grinding efficiency. The size range of the popcorn should be between 0.3 and 0.5 cm in diameter. Please click here to view a larger version of this figure.

2. Cryogrinding

1. To start, ensure that there is enough LN₂ available to chill the freezer mill, grinding vials, supplies and to operate the machine for all samples. For less than five samples, 30-35 L of LN₂ should suffice. Fill the freezer mill chamber with LN₂ up to the fill line.
   WARNING: All steps involving LN₂ should be performed in a well-ventilated area and the freezer mill itself should be located in such an area to avoid risk of asphyxiation. Wear personal protective equipment including proper footwear, lab coat, safety glasses, a base layer of nitrile gloves, then thermal gloves, followed by another pair of nitrile gloves. Use extreme caution when handling LN₂.
2. Close the freezer mill lid slowly, avoiding the splash of LN₂ and allow a few min for the machine to cool down. It may be necessary to refill the chamber with LN₂ to compensate for the loss due to evaporation before proceeding to the next step.
   NOTE: Refill LN₂ as needed only up to the fill line. Do not overfill the chamber as this can be both dangerous and detrimental to the machine. Automated refill systems are available for freezer mill that can replenish the evaporating LN2 as needed directly from a storage tank connected to the freezer mill.
3. Pre-chill the large grinding vials and the magnetic impactor bar by dunking them in LN₂ kept in a separate small dewar. Decant all the LN₂ when the LN₂ stops bubbling. Then add the sample/yeast popcorn to the grinding vial and seal it tightly with the two stainless steel end plugs.
   1. Do not fill more than one-third of the grinding vial with the sample as that can reduce the efficiency of grinding. Instead, larger samples can be processed by dividing them into two or more vials and grinding them sequentially. Smaller grinding vials can be used for smaller samples (up to 3 mL).
4. Place the grinding vial in the freezer mill chamber and lock it in place. Close the lid.
5. For budding yeast, grind the samples for a total of three cycles for 2 min/cycle (with 2 min break for cooling between cycles) at a crushing rate of 14. When the machine stops after completing the cycles, open the freezer mill lid slowly and carefully unlock the vial with the powdered frozen cell lysate and remove it from the freezer mill. If multiple small vials are used, work quickly to remove one vial at a time and place them in dry ice.
   NOTE: There is usually no need to adjust the grinding parameters when using vials of different sizes with the same type of sample. However, the grinding parameters such as the number of cycles and the grinding rate need to be empirically determined for different sample types, depending on the ease with which they lyse. Most mammalian cells and soft tissues will lyse in 1-2 cycles at a crushing rate of 10. Harder to lyse samples such as bacteria, yeast, fly larvae and adult fruit flies require 3-6 cycles at the maximum rate, while hard tissues such as bone, teeth, etc. may require up to 10 cycles. A small aliquot of the thawed sample can be viewed under the microscope before and after grinding to count the number of intact, unlysed cells to determine the lysis efficiency. An excellent analysis of the impact of cryogrinding parameters on the release of proteins and DNA from budding yeast has been published previously¹⁵.
6. Working quickly so as not to allow the sample to thaw out, carefully unscrew one of the end pieces (pick the one that appears to have become somewhat loose during the grinding) using the opening tool. Then, use a pair of long forceps (that are pre-chilled in a bucket with dry ice) to remove the impactor bar. Collect the pulverized sample by inverting and tapping the grinding vial onto a polystyrene weighing dish pre-chilled with LN₂ and kept on dry ice.
7. Once all of the powdered frozen cell lysate has been recovered from the grinding vial, pour the powdered lysate from the weighing dish back into the 50 mL tube and proceed immediately to the slow thawing step described next for best results. Alternatively, store the frozen powdered lysate overnight at -80 °C, although this may result in some degradation.
8. Prepare an ice bucket with a 50% slurry of ice and water and place it on a stir plate with a magnetic stirrer. Submerge a wire rack or another suitable rack in the ice slurry to hold the samples.
   1. Then, slowly thaw the samples on an ice slurry bath with constant agitation of the slurry using a magnetic stirrer. Add more ice to replace the melting ice (some water may need to be discarded to prevent the ice bucket from overflowing). Since several inhibitors have short half lives in aqueous buffers, add additional protease inhibitor cocktail (see Table of Materials) and 10 μM proteasome inhibitor MG-132 to the lysate once the samples start thawing (after approximately 30 min).
   2. Remove ice formed on the outside of the tubes every 5 min to expedite the thawing process. Note that rapid thawing of the samples at room temperature or higher can lead to significant degradation.
9. After the samples have thawed completely (it may take well over an hour depending on the amount of sample), centrifuge the lysate at ~3,220 g for 20 min in a refrigerated tabletop centrifuge at 4 °C to remove the bulk of the cell debris from the lysate.
10. Transfer the supernatant to 50 mL polycarbonate centrifuge tubes that have been pre-chilled on ice and centrifuge the samples at 16,000 g for 20 min at 4 °C (see Table of Materials).
11. Transfer only the clear supernatant consisting of the clarified whole cell extract from the center of the liquid column in the tube very slowly and carefully, without disturbing the cloudy lipid layer (Figure 2), into a chilled 15 mL tube using pre-chilled serological pipettes. Avoid removing all of the lysate in the centrifuge tube to prevent carryover of lipids and debris. The leftover lysate in the centrifuge tube can be centrifuged multiple times to recover small amounts of extract after each spin.
    ​NOTE: Lipids along with any denatured proteins would appear cloudy/milky and are located at the top of the liquid/air interface, and occasionally above the pellet itself that consists of insoluble cellular debris (Figure 2).

Figure 2: Extract layers in a centrifuge tube. The major visible features of the whole cell extract in a tube following centrifugation at 16,000 g for 20 min are indicated. The relative abundance of each feature depends on the sample type, the growth phase of the cells (exponential versus stationary), the amount of lysis buffer used to resuspend cells and the lysis efficiency. Please click here to view a larger version of this figure.

12. Centrifuge any remaining lysate for 5 min to recover more of the extract. Repeat this 5 min centrifugation multiple times if needed to recover as much of the clear extract as possible. Discard the cloudy lysate containing lipids close to the bottom of the tube near the pellet.
13. Use these extracts for downstream applications such as protein complex purification, immunoprecipitation and proteomic analysis.

Results

We compared two different methods for yeast cell lysis, namely glass bead milling at 4 °C and an automated cryogrinding method at -196 °C, to assess the relative recovery proteins in the cell extracts prepared with both methods. For this study, we chose to use a budding yeast strain YAG 1177 (MAT a lys2-810 leu2-3,-112 ura3-52 his3-Δ200 trp1-1[am] ubi1-Δ1::TRP1 ubi2-Δ2::ura3 ubi3-Δub-2 ubi4-Δ2::LEU2 [pUB39] [pUB221])¹⁶ carrying a ...

Discussion

A limitation of studying native proteins from yeast is the inefficient lysis of yeast cells due to their tough cell wall. Although several methods have been developed, the most consistent and efficient method in our hands is the cryogrinding of yeast cells flash frozen as popcorn. This method allows the reliable preparation of high-quality whole cell extracts from budding yeast compared to other lysis methods. The representative results demonstrated that cryogrinding is superior to a popular mechanical method for ye...

Materials

Name	Company	Catalog Number	Comments
50 mL polycarbonate tubes with screw caps	Beckman	357002	Centrifuge tubes
BD Bacto Peptone	BD Biosiences	211677	Yeast YPD media component
BD Bacto Yeast Extract	BD Biosiences	212750	Yeast YPD media component
Beckman Avanti centrifuge	Beckman	B38624	High speed centrifuge
Beckman JLA-9.1000	Beckman	366754	Rotor
D-(+)-Dextrose Anhydrous	MP Biomedicals	901521	Yeast YPD media component
Eppendorf A-4-44	Eppendorf	22637461	Swinging bucket rotor
Eppendorf refrigerated centrifuge 5810 R	Eppendorf	22625101	Refrigerated centrifuge
Glycerol	SIGMA-ALDRICH	G5150-1GA	Volume excluder and cryoprotectant
HEPES	FisherBiotech	BP310-100	Buffer
HIS6 antibody	Novagen	70796	Antibody for HIS tag
KCl	SIGMA-ALDRICH	P9541-1KG	Salt for maintaining ionic strength
MG-132	CALBIOCHEM	474790	Proteasome Inhibitor
Phosphatase inhibitor cocktail	ThermoFisher Scientific	A32957	Phosphatase inhibitor cocktail
Ponceau S	SIGMA	P7170-1L	Protein Stain
Protease inhibitor cocktail	ThermoFisher Scientific	A32963	Protease inhibitor cocktail
Rotor JLA 25.500	Beckman	JLA 25.500	Rotor
Sodium Butyrate	EM Science	BX2165-1	Histone Deacetylase Inhibitor
Sodium Fluoride	Sigma-Aldrich	S6521	Phosphatase Inhibitor
Sodium Vanadate	MP Biomedicals	159664	Phosphatase Inhibitor
Sodium β-glycerophosphate	Alfa Aesar	13408-09-8	Phosphatase Inhibitor
Spex Certiprep 6850 freezer mill	SPEX Sample Prep	6850	Freezer Mill
TALON Metal Affinity Resin	BD Biosiences	635502	For pulling down HIS tagged proteins
Tween 20	VWR International	VW1521-07	Non-ionic detergent
β-Mercaptoethanol	AMRESCO	M131-250ML	Reducing agent