The overall goal of this procedure is to purify ribosomes from mitochondria of human cell lines on a large scale that is sufficient for structural studies. This mitochondrial ribosome purification method enables the characterization of translating complexes, mutants, quality control assemblies, and mitoribosomal subunit intermediates. These can be used to answer key questions about protein synthesis in mitochondria.
The main advantage of this technique is that nitrogen cavitation is used for cell lysis and only 10 to the 10 culture cells are needed to prepare mitoribosomes for high resolution structure determination. This technique can result in the discovery of new components of the mitochondria translation machinery and can be further used for the development of novel therapeutics for cancer treatment. After culturing HEK293S cells according to the text protocol, harvest two one liter tubes of cells by centrifugation at 1, 000 times g and four degrees Celsius for seven minutes.
Carefully decant the supernatant and quickly resuspend each pellet in 100 milliliters of PBS. Then, pool the cells and centrifuge the suspension at 1, 200 times g and four degrees Celsius for 10 minutes. Next, after carefully decanting the supernatant, weigh the pellet.
Then, use 120 milliliters of MIB buffer to resuspend it and leave the resuspended cells to swell in the cold room for 20 minutes. Transfer the swelled cells to a pre-cooled nitrogen cavitation chamber on ice. Then, add 45 milliliters of SM4 buffer to the cells.
Fasten the nitrogen cavitation chamber and fill it with nitrogen until the pressure reaches 500 PSI. Then, close the taps and keep it on ice for 20 minutes. The nitrogen cavitation cell lysis method prevents external physical stress on the cells, avoids heat damage to organelles, and the nitrogen gas protects the cells from oxidation.
In addition, it's a highly reproducible method. Slowly release the pressure in the chamber and collect the lysate. Then, to remove the cell debris and nuclei, centrifuge the lysed material at 800 times g and four degrees Celsius for 10 minutes.
Collect the supernatant by pouring it through a folded piece of muslin cloth into a beaker kept on ice. Do not discard the pellet. Resuspend the pellet in 90 milliliters of MiBSM buffer.
Then, using a Teflon glass down homogenizer, homogenize the sample and repeat the centrifugation at 800 times g and four degrees Celsius for 10 minutes. Again, collect the supernatant by pouring it through a folded piece of muslin cloth into a beaker kept on ice. Then, combine this supernatant with the first supernatant.
Centrifuge the sample at 1, 000 times g and four degrees celsius for 15 minutes. Then, collect the supernatant as before. After discarding the pellet, centrifuge the supernatant containing crude mitochondria at 10, 000 times g and four degrees Celsius for 15 minutes.
Carefully wash out any loose pellet without disturbing the tight portion. Then, use 10 milliliters of MiBSM buffer to resuspend the tight pellet. Add 200 units of RNase-Free DNase to the sample to remove the genomic DNA and rotate the tube on a roller in the cold room for 20 minutes to evenly digest the sample.
Centrifuge the sample at 10, 000 times g and four degrees celsius for 15 minutes and use two milliliters of SEM buffer to resuspend the pellet. Load the entire mitochondrial suspension on top of the sucrose gradient. After spinning the gradient according to the text protocol, using a transfer pipette, carefully collect the brown band migrating at the interface of 32%and 60%sucrose.
This protocol uses nitrogen cavitation to break the cells. Once mastered, a large scale isolation of highly pure, intact mitochondria can be done within nine hours and can easily be modified and adapted for different cell types and scales. After defrosting frozen mitochondria on ice, add two volumes of lysis buffer to three milliliters of mitochondria.
Immediately mix by inverting the tube several times. Use a small Teflon glass down homogenizer to assist with lysis and incubate the homogenate on ice for five to 10 minutes to complete the reaction. Centrifuge the lysate at 30, 000 times g and four degrees Celsius for 20 minutes to remove the insoluble material.
Then, carefully collect the supernatant and discard the pellet. Repeat the centrifugation to ensure clarification of the supernatant. Then, carefully collect the supernatant and discard the pellet.
For every milliliter of lysed material, prepare a sucrose cushion in a TLA-120.2 ultra clear tube by adding 0.4 milliliters of sucrose cushion to the tubes. Layer approximately one milliliter of lysed mitochondria onto each sucrose cushion resulting in a lysate-to-cushion ratio of 2.5 to one. Then, centrifuge the sample in a TLA-120.2 rotor at 231, 550 times g and four degrees Celsius for 45 minutes.
Discard the supernatant and use 100 microliters of the resuspension buffer to rinse the tube and remove residual sucrose. Resuspend the pellets and combine them in a total of 100 microliters of resuspension buffer. While resuspending the cushion pellet, it is essential to perform on ice to avoid heating of the sample and also to avoid foaming of the sample in order to ensure the structural integrity of the mitochondrial ribosomes.
Vortex the sample on slow speed for 30 seconds to dissolve the remaining aggregates and centrifuge the tubes at 17, 949 times g and four degrees Celsius for 10 minutes. Carefully collect the supernatant and repeat the centrifugation. After measuring mitoribosome absorption at A260, load the entire sample onto a single linear sucrose gradient tube.
Centrifuge the sample in a TLS-55 rotor at 213, 626 times g and four degrees Celsius for 120 minutes. To fraction the gradient, puncture the bottom of the tube and collect the sample drop-wise in a 96-well plate. Mitoribosome purification should not take more than seven hours for a sufficient amount of mitoribosomes.
As demonstrated in this discontinuous sucrose gradient, the purified mitochondria migrate to the brown lower band at the 32-60%interface. Following the separation of mitoribosomes from soluble mitochondrial complexes on a sucrose gradient, two major mitoribosomal populations are identified, the monosome 55S and large subunit 39S. The presence of the large subunit fraction suggests that cells are harvested in an actively dividing state.
This panel shows a micrograph with the sample from monosome peak two at a calibrated magnification of 1.23 angstroms per pixel. Shown here are data after initial processing revealing intact monosomes. Finally, a monosome 3D reconstruction is illustrated in this panel.
After watching this video, you should have a good understanding of how to prepare intact human mitoribosomes for structural and biochemical studies. Our protocol offers high quality final preparations that could be extrapolated to other mitochondrial macromolecules.
