Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

Yeast is popular model organism, not just for genetic studies but also for biochemical studies of proteins and other macromolecules extracted from wild type and mutant yeast strains. However, due to their tough cell walls, a major challenge that researchers face is this efficient lysis of yeast cells without damaging the cellular content. The isolation of intact biological macromolecules is usually critical, dependent on temperature.

Preparing the extracts at low temperature ensures that the intracellular proteases and nucleases stay inactive, resulting in reliable isolation of intact protein, nucleic acids, and other macromolecules for sufficient lysis. Various methods are available for obtaining yeast extracts through enzymatic, chemical and physical lysis. However enzymatic lysis is expensive due to the requirement of purified enzymes to digest the cell wall, thus limiting the use of smaller number of cells.

Further the enzymatic reactions has to be monitored closely to prevent over digestion and premature lysis of the cells. Chemical methods using strong denaturing agents, though effective, result in the denaturation of proteins and as such, are not suitable for the isolation of protein complexes or functional enzymes. Physical methods, such as lysis under high pressure in a French Press can be carried out in the cold at four Celsius but that is not cold enough to prevent degradation and denaturation of all native proteins.

Other popular physical methods are the use of a mortar and pestle, blender, or coffee grinder to grind yeast cells resuspended in a lysis buffer and frozen as droplets or grinding and mixture of glass beads and yeast in a fast prep bead beater mill or a lysis by sonication. However, manual grinding is labor intensive and the resulting protein yield can vary considerably depending on the techniques employed while lysis by sonication or grinding in a blender, coffee grinder, or bead beater mill generates a substantial amount of heat, which results in a protein denaturation and degradation. Therefore, to obtain yeast cell lysis with proteins in their native state with minimal denaturation and degradation for applications like purifications of functional protein complexes, biochemical assays or detection of fleeting post-translational modifications, it is essential to use an easily scalable lysis method that will minimize heat generation while allowing for efficient lysis regardless of the quantity of cells.

Here we describe a method that involves the use of a cryo freezer mill for the lysis of cells in liquid nitrogen at minus 196 Celsius to generate frozen cell extracts thereby allowing the recovery of intact functional macromolecules, such as proteins or DNA and RNA, at a very low temperatures which minimizes denaturation and degradation. This method can be readily adapted for use with any type of cell or tissue sample from any species, including forensic and even ancient samples recovered from archeological sites or found frozen in permafrost. The freezer mill uses a superconducting electromagnetic grinding chamber that rapidly moves a solid metal bar back and forth within a polycarbonate file containing the sample to be pulverized between stainless steel end plugs.

This physical cell lysis method allows for a more efficient lysis and reproducibly results in a better quality extract. Yeast cells were grown at a concentration of 10 million cells per mil. These yeast cells were placed pre chilled centrifuge bottle and were spun for 10 minutes at 2, 400 G.Once the spin is complete, decant the supernatant without disturbing the pelleted cells.

This pellet was resuspended in a small amount of ice cold water, about half the initial volume of the culture to wash the cells. The resuspended cells were again spun down for another 10 minutes at 2, 400 G.Once the next spin is complete, we, again, decant all the supernatant without disturbing the pellet. This pellet will then be resuspended and 15 ML of ice cold lysis buffer.

Ensure that the pellet is completely resuspended. The resuspended cells should remain on ice in a pre-marked tube until ready for the next step. Always wear personal protective equipment when handling liquid nitrogen, such as safety glasses, hand protection, and lab coats.

To maximize our dexterity, we often wear a pair of white thermal gloves sandwiched between two pairs of nitrile gloves. Change the outer and nitrile gloves frequently, as they rip easily after being exposed to liquid nitrogen. In our lab, we transfer an aliquot of liquid nitrogen into a small five liter liquid nitrogen container.

Liquid nitrogen is then poured into a 50 ML tube that has been pre chilled on dry ice. Once the 50 ML tube is full of liquid nitrogen, we add the yeast extract slowly, in a drop wise manner to the tube in 1.5 milliliter increments. To minimize the formation of large popcorn pellets, the yeast suspension is added in a circular motion.

To further prevent the formation of large popcorn pellets, we use large forceps to ensure all pellets are between 0.3 and 0.5 centimeters in diameter. Because liquid nitrogen is always evaporating, be sure to refill the tube with liquid nitrogen before adding each 1.5 mil aliquot of yeast suspension. Once all of the yeast suspension has been made into popcorn allow the tube with popcorn to sit without the lid for two to three minutes to let all of the residual liquid nitrogen to evaporate.

To be sure that all of the liquid nitrogen has evaporated, close the lid nearly completely and shake gently. If there is a hissing noise, it indicates that the liquid nitrogen has not yet fully evaporated. Open the lid for another minute or so to allow the remaining liquid nitrogen to evaporate before closing the lid tightly.

Residual liquid in the tube may cause an explosion if the lid is closed prematurely. This yeast popcorn can be stored almost indefinitely in minus 80 degrees C.For at least five samples, ensure that you have 30 to 35 liters of liquid nitrogen available. Fill the freezer mill to the full line and close the lid to allow the freezer mill to pre chill for a few minutes.

Again, always remember to wear personal protective gear when dealing with liquid nitrogen. The polycarbonate grinding vials, stainless steel end plugs, and impactor bar must be pre chilled with liquid nitrogen. Keep these components submerged until the liquid nitrogen stops bubbling.

Remember to keep all popcorn samples on dry ice until the entire grinding process is complete. Pull out all the liquid nitrogen from the grinding vials and transfer your popcorn into the vial. Don't forget to place a magnetic impactor bar in the grinding vial as well.

Seal the grinding vial by carefully placing the stainless steel plug evenly on the end of the grinding vial. We bang each end of the grinding vial on a wooden breadboard with a vibration absorbing rubber backing to ensure that the end plugs are placed correctly and are sealing the grinding vials tightly. It is important to secure the end plugs to ensure that they will not come loose and allow the samples to spill in the freezer mill during the grinding process.

Place a grinding vial in the freezer mill grinding chamber and lock in place. Close the freezer mill lid and grind the sample for three cycles at two minutes per cycle, at a crushing rate of 14. After the grinding cycles are complete unlock the vial with the frozen powder cell lysate.

To prevent the lysate from thawing out, work quickly and carefully to unscrew one of the end plugs using the opening tool. Once the vial is open, remove the impactor bar and quickly transfer the frozen powder extract on a pre chilled plastic weighing dish. Recover as much of the frozen extract as possible by banging the vial on the wooden breadboard.

Once all the powder is removed from the vial, quickly transfer all of the powder extracts into a pre-labeled 15 mil tube kept on dry ice. Although it is best to proceed with thawing and using the extracts right away to minimize protein degradation, the frozen powder lysate can be stored overnight at minus 80, if needed. Thaw out the powder lysate slowly in an ice slurry that is continuously circulated using a magnetic stir bar in an ice bucket.

To facilitate even thawing, remove ice that develops on the outside of the tubes frequently, about every five minutes or so. Once the extracts begin to thaw at 1X protease inhibitor cocktail and 10 micromolar proteasome inhibitor MG132 to prevent protein degradation. Once the samples are completely thawed, which can take over an hour, spin down the samples at 3, 220 G for 20 minutes at four Celsius degree.

This spin will remove most of the cell debris from the lysate. When the spin is complete transfer the supernatant into polycarbonate bottles that have been pre chilled on ice and discard the pellet. Centrifuge the supernate and samples at 16, 000 G at four Celsius degree for 20 minutes to clarify the extract.

After the spin is complete, recover only the clear supernatant carefully from the middle of the liquid column without disturbing the cloudy layer containing the piece on top or the pelleted debris at the bottom of the centrifuge tube. Transfer the clear lysate into a fresh pre chilled 15 Mil tube. The remaining cloudy liquid can be recentrifuged for five minutes at the same speed to allow the recovery of some more of the wholesale lysate.

The extracts are now ready for use in experiments, such as purification of protein complexes and immunoprecipitation. We compared two different methods of yeast cell lysis, namely glass bead milling at four Celsius and an automated cryo grinding method at minus 196 Celsius to assess the relative recovered proteins and cell extracts prepared with both methods. From this study we chose to use a budding yeast strain carrying a high copy plasmid expressing a tandem HIS-MYC tagged ubiquitin.

So we can assess the affinity of recovery of tagged polyubiquitinated proteins from wholesale extracts, following affinity purification using TALON cobalt HIS tag affinity beads followed by Western blotting with a monoclonal HIS tag antibody. Polyubiquitinated proteins that are normally very short-lived due to their rapid degradation by the ubiquitin proteasome machinery and thus offer a very good way to compare the quality of wholesale extracts prepared by different methods. As we see by the right half of the Ponceau stained membrane, we recovered a higher total protein yield in the wholesale extracts prepared by the cryo freezer mill protocol as judged by the more intensive Ponceau staining throughout the wholesale extract lane.

This is especially clear in the lower and upper parts of the freezer mill whole extracts lanes. Additionally, as shown by the right half of the HIS tag Western blot, we observed a better recovery of HIS-MYC tag ubiquitinated protein followed by pull down using Talon beads from the wholesale extracts prepared by cryogenic grinding. We conclude that the cryogenic freezer mill is superior to other methods for the preparation of cell extracts, especially when functional macromolecules are required for downstream application.