Fast Parallel Proteolysis
Our featured method this month is Fast Parallel Proteolysis, which is performed in cell biology labs to determine the thermostability of proteins that are extracted from cells. Thermostability refers to a protein’s ability to resist physical and chemical changes occurring at high temperatures. Fast parallel proteolysis is a relatively new method that was first proposed in 2012 by a group of scientists in the Netherlands. It has many biochemical applications as it can be used to determine the stability of proteins under varying conditions other than temperature, such as pH and the presence of chemicals. It even has applications in the field of genetics, where it can be used to show how exactly a specific mutation affects the structure of a protein.
To begin the process, we first need to remove our protein of interest from the cells of our sample. To accomplish this, we need to start by breaking apart the membrane of a cell by performing cell lysis. There are several ways to perform cell lysis, which are classified as either mechanical or non-mechanical. Mechanical methods involve using high pressure or fast-moving glass beads to physically tear holes in the cell membrane, while non-mechanical methods usually involve the use of enzymes or alkaline solutions to disrupt the bonds of the phospholipids that make up the membrane. Once the cell is broken open, we can access the proteins inside, which are collectively called cell lysate.
Next, we must isolate our protein of interest from the thousands of others that make up the cell lysate. We can do this by marking the protein using an intrinsically disordered peptide tag. To put this word into simpler terms, remember that a peptide is a short chain of amino acids (the building blocks of protein) linked by special chemical bonds known as peptide bonds. These peptides usually form a larger, ordered protein structure through folding due to interactions with other molecules. However, peptides are considered intrinsically disordered if they are unfolded and lack an organized 3D structure. By tagging our target protein with these molecules, they will not aggregate, or cluster with, neighboring compounds extracted from the cell.
Once our protein of interest has been isolated and purified from the others in the sample, it will be mixed with an enzyme called a protease and divided into several different tubes. The function of a protease is to speed up reactions that break apart proteins into smaller pieces. It is important for this step to occur in a very cold environment so degradation of the proteins does not occur yet, with 4°C (39.2°F) being the preferable temperature.
The mixtures will then be placed into a device called a gradient thermal cycler, which is an instrument that raises and lowers the temperature of a sample in discrete, pre-programmed steps. For each mixture, a different maximal temperature should be reached, with the lowest maximum temperature preferably reaching 40 °C (104 °F) and the highest maximum temperature reaching no more than 85 °C (185 °F).
Once the gradient thermal cycler lowers the temperature back down to 4°C, the reaction products will be analyzed using a gel-based method such as SDS-PAGE or western blot. (If you are not familiar with either of these, please click on the highlighted links for earlier issues of Eta Zeta’s Method of the Month where both were covered!)
In both methods, the reaction products from each tube will be placed into a separate well in the gel, so we are able to visualize how well the protein can be detected for each sample and compared to a known control that is very heat stable. The wells should be ordered by increasing temperature for the best results.
After a certain temperature, we will notice that we will no longer be able to visualize the band representing our target protein because it has been degraded. For a great example of what this looks like on a gel, please check out Figure 7 of this article! The higher the temperature that the sample was able to reach before it degraded and could no longer be visualized on the gel, the more thermostable the protein was considered to be.
In summary, the steps of fast parallel proteolysis are as follows: cell lysis, protein isolation, addition of a protease, thermal cycling, and visualization of results. We hope you enjoyed this feature, and make sure to read the rest of the Eta Zeta Biology Journal’s Method of the Month series!
