The overall goal of this procedure is to purify intact, highly active yeast ribosomes. This is accomplished by first lysing the cells and clarifying the lysate by high speed centrifugation. To obtain the S 30 defraction, a cysteine charged SFO link column is then used to purify the ribosomes.
This is followed by removal of contaminating tRNAs with pur mycin treatment as a final purification step. The ribosomes are subject to high speed ultra centrification. The main advantage of this technique over existing methods like ultra centrification purification, is that use of the sulfa link column rapidly separates ribosomes from contaminating exonuclease and proteases.
Further, the pur mycin treatment step separates ribosomes from contaminating RNA species such as tRNAs. These steps ensure production of large yields of highly active, intact purified ribosomes for use in biochemical and structural studies. Demonstrating the procedure will be Jonathan Leschen, a postdoctoral research associate from my laboratory.
Begin this protocol by pipetting a 50%slurry of sulfa link coupling gel as supplied by the manufacturer into two 10 milliliter plastic vials with five milliliters distributed into each vial. After briefly centrifuging the vials at 850 times G.Carefully remove the supernatants. Wash the gel three times in coupling buffer by resus, suspending the beads in five milliliters, and then centrifuging briefly at 850 times G.Remove the supernatant by pipette After washing the resin, add five milliliters of a 50 millimolar solution of L cysteine in coupling buffer to each tube.
Mix the slurry for one hour at 25 degrees Celsius. Then remove the residual L cysteine by washing the resin three times with coupling buffer alone. Next, resuspend the gel in 10 milliliters of binding buffer and equilibrate the resin by washing it three times.
Lastly, decant the resin into a 10 milliliter centrifuge column and cap it. The resin can be stored at four degrees Celsius and has a resin capacity of approximately 20 a two 60 units per milliliter during the chromatographic purification of ribosomes. Using a cystine charged sulfa link resin.
Maintain all components on ice and prepare all solutions using RNAs free water and sterile filtration. Grow yeast cells overnight to mid log phase in the appropriate media. After paling the cells by centrifugation, wash one gram of cells in binding buffer and centrifuge again, then resuspend the cells in one milliliter of binding buffer to an approximate total volume of two milliliters.
Disrupt the cells using an equal volume of 0.5 millimeter glass beads chilled to four degrees Celsius, as well as a mini bead beader. Remove unbroken cells, organelles, and cellular debris by centrifugation at 30, 000 times, G for 30 minutes in an ultracentrifuge. Meanwhile, pellet the resin for one minute at 1000 times G.To remove the storage solution immediately before use.
Two milliliters of resin are used for every one gram of cells. Following ultracentrifugation of the samples, remove the cell lysate supernatants from the 30, 000 times G spin termed S 30. Take care to minimize contamination from either the lipid fraction at the very top or the cell debris at the bottom of the tubes.
Transfer the supernatants directly into the charged SFO link slurry. Incubate the S 30 SFO link mixture on ice for 15 minutes. Place the columns into 50 milliliter conical tubes and centrifuge at 1000 times G for one minute, transfer the flow through fractions back into the columns, cap the columns and invert them to mix.
Incubate the samples for a further 15 minutes on ice. Then place the columns into 50 milliliter conical tubes and centrifuge at 1000 times G For one minute, discard the flow through. After adding five milliliters of binding buffer, cap the columns and invert to mix until resuspended.
Remove the caps and centrifuge the columns at 1000 times G For one minute. Repeat this washing protocol and additional two times. Next, add 1.5 milliliters of elution buffer to each column.
Mix the slurries by hand and incubate on ice for two minutes. As a final step, place the columns into new 50 milliliter conical tubes and centrifuge at 1000 times G.For one minute, collect eluate and repeat the elucian step once more so that the final combined sample volumes are approximately three milliliters. In order to strip the ribosomes from endogenous peptol, T-R-N-A-A treatment with pur mycin is performed.
First, neutralize a 100 millimolar pur mycin solution to pH 7.5 by adding one molar potassium hydroxide dropwise. Add room temperature. Add the neutralized pur mycin solution to approximately one millimolar final concentration in the eluate.
Then add 100 millimolar GTP to a final concentration of one millimolar. Incubate the mixture for 30 minutes at 30 degrees Celsius. Following incubation, place one milliliter of cushion buffer into a four milliliter volume polycarbonate ultracentrifuge tube.
Gently layer the pur mycin treated elution fractions on top of the cushion and centrifuge the samples at 100, 000 times G overnight. After aspirating the supernatants wash the pellets containing the purified ribosomes with one milliliter of cushion buffer. Repeat this wash with a second milliliter of cushion buffer.
Then Reza spend the ribosomes in 100 microliters of cushion buffer by gentle disruption using a glass rod. Next, cover the tubes with param and shake at a moderate speed in a cold room vortex. After one hour of shaking, transfer the contents to a micro centrifuge tube centrifuge for five minutes using maximum speed at four degrees Celsius.
Transfer the supernatants to fresh tubes. Layer the supernatants on top of two milliliters of cushion buffer in an ultra centrifuge tube, followed by centrifugation at 100, 000 times G.Overnight. The next day, remove the tubes from the centrifuge rotor and aspirate supernatants.
Using the method just described. Wash the pellets and resuspend the ribosomes with storage buffer. Finally, quantify the purified ribosomes spectra photometrically before storing at minus 80 degrees Celsius.
Shown here is an example of RNA species extracted from each of the three major steps of the protocol, including the total cell lysates, the sulf olink, purified ribosomes, and the sufu link purified ribosomes subsequently treated with pur mycin and sedimented through a glycerol cushion. RNA size markers are also shown while our RNAs are the major species present in total cell lysates. These also contain a large number of other RNA species.
Ribosomes purified from the sulfa link column contain a large amount of tRNAs due to the high affinity of the column bed for these species. Treatment of this fraction with piro mycin results in hydrolysis of peptides from peptol tRNAs, and promotes dissociation of these species from ribosomes. The pure mycin treated samples lack co purifying TRNA species and thus represent completely pure ribosomes.
As reported previously, these ribosomes are highly intact and biochemically active, making them ideal substrates for detailed functional and structural analyses. Following this procedure, other methods like assays to monitor binding of ligands to ribosomes and structural analysis can be performed in order to answer additional questions like, what are the biochemical and biophysical properties of eukaryotic ribosomes.
