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)

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,....

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 high copy plasmid expressing a tand.......

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

List of materials used in this article

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