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.
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.
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 cryogenic2,11. 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 science12,13, 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.
1. Preparation of Yeast Popcorn
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
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.
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 high copy plasmid expressing a tandem HIS-MYC tagged Ubiquitin (HIS-MYC-Ub) so we can assess the efficiency of recovery of tagged ubiquitinated proteins from extracts following affinity purification.
We tested a quick protocol that uses glass bead milling to lyse yeast cells9 at 4 °C and the cryogrinding method using frozen yeast cells (yeast popcorn) and grinding them in LN2 in a freezer mill. We report a clear difference with the two methods of extract preparation.
In Figure 3A we demonstrate that we can achieve a decidedly higher total protein yield from whole cell extracts (WCE) prepared using freezer grinding as determined by Ponceau S staining after loading equivalent amounts of WCE prepared from the same number of cells using the two different extract preparation methods. Further, Figure 3B shows higher recovery of HIS-MYC tagged ubiquitylated proteins following pull down using Talon beads from the WCE prepared by cryogrinding, when compared to the WCE prepared using the bead beater, despite using extracts prepared from the same number of cells in both cases.
Figure 3: Cryogrinding leads to higher total protein yields in cell extracts and better recovery of tagged proteins following pull down of proteins from these extracts. (A) Whole cell extracts (WCE) were prepared as described in this protocol for the cryogrinding or as previously described for the bead milling method.9 Five hundred mL of the yeast strain carrying HIS-MYC-Ub was grown to density of 107 cells/mL. Then 250 mL of the culture was used in parallel to prepare extracts by each method. Equivalent amounts of WCE was prepared from the same number of cells using the two different methods. HIS tagged proteins were then pulled down using Talon beads (IP) from the same amount of each extract. Finally, these affinity purified proteins were compared by resolving them on a denaturing polyacrylamide gel and transferring to a nitrocellulose membrane followed by staining with Ponceau S. Notice the stronger staining throughout the lane containing WCE prepared using the freezer grinding method. (B) The Ponceau S stained nitrocellulose membrane shown in (A) was then processed for Western blotting using HIS tag antibodies to compare the amounts of HIS-Ub tagged proteins pulled down from equal amounts of extract prepared by the two methods. Several bands were clearly overrepresented in the pulldown using the WCE prepared by cryogrinding (indicated by the red arrows), while the corresponding bands in the pulldown using WCE prepared by bead beating were either considerably weaker in intensity, or undetectable. Please click here to view a larger version of this figure.
Overall, the representative results show that cryogrinding of yeast popcorn leads to higher yields and superior protein isolation based on the stronger Ponceau S stained protein bands in the WCE prepared by cryogrinding (Figure 3A). This is likely due to more efficient lysis and limited protein degradation in the extract prepared by cryogrinding, as indicated by the presence of stronger bands in the Western blot lane showing HIS-tagged proteins pulled down (IP) from the cryogenic extract (Figure 3B). While the glass bead milling protocol is faster and less labor intensive, it may not be suitable for proteins that are temperature sensitive or highly unstable. We also find that the isolation of posttranslational modifications such as polyubiquitinated proteins is more efficient with the cryogrinding protocol16,17,18.
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 yeast lysis that employs bead milling at 4 °C (Figure 3A and 3B). Cryogrinding efficiently lyses yeast cells while the samples are processed at a temperature of -196 °C in a LN2 bath, therefore maintaining protein integrity by greatly limiting heat induced protein denaturation and degradation that occurs with many other methods of extract preparation. This allows for the analysis of highly labile macromolecules in the samples and permits the purification of functional protein complexes. Further, the use of the freezer mill also replaces the highly labor-intensive traditional manual grinding of yeast popcorn with a mortar, allowing for faster and efficient sample processing. Finally, the achievement of yeast cell lysis without the use of any expensive enzymes provides for significant savings in the long run.
The major downside of a freezer mill is the upfront cost involved in purchasing the equipment, as well as the minimal maintenance and running costs (i.e., large quantities of liquid nitrogen) involved. It also requires careful handling of potentially dangerous LN2 for both the preparation of yeast popcorn and the cryogrinding steps. Larger samples need to be processed individually, which may become time consuming if multiple samples need to be processed. In addition, when processing multiple samples, the grinding vials, caps and impactor bars must be properly washed and wiped clean before re-use (alternatively, several sets of spare accessories must be maintained).
The protocol demonstrated here is valuable for the preparation of yeast protein samples for protein affinity purification, immunoprecipitation studies, sample preparation for Western blotting for labile proteins or their modifications, or for proteomic analyses16,19. In addition, cryogrinding of the yeast popcorn can also be utilized for sheared genomic DNA13,20,21 or RNA22,23 isolation while minimizing nuclease activity, thus ensuring sample preservation24. This versatile method can be used as is for most microorganisms such as bacteria21,22 and yeasts15, and can be quickly adapted for mammalian cells, both plant25 and animal26 tissues, food products27, marine specimens21,28 including corals23, forensic samples20 including hair29 and even fossils30 as well as the remains of extinct lifeforms frozen in permafrost31.
In conclusion, cryogrinding in a freezer mill is an efficient method for protein and other macromolecule isolation from a variety of samples derived from diverse sources for multiple downstream biochemical applications.
The authors have nothing to disclose.
Research in the Gunjan lab is supported by funding from the National Institutes of Health, National Science Foundation and the Florida Department of Health. We thank undergraduate student John Parker for technical assistance.
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 |