Protein purification refers to the characterization of proteins in order to use them for certain laboratory or medical functions. It also includes the separation of protein and non-protein substances in a cell so that the needed protein is isolated for further use.
What is Protein Purification?
Protein purification is the process where the needed kind of protein is isolated and extracted from a mixture of cells, tissues, and organisms for either preparative or analytical use.
Preparative use means that a protein (or large quantities of the same protein) is taken so that it can be used for commercial or medical purposes. Take, for example, insulin. It goes through protein purification so that it can be used for those who are diabetic.
On the other hand, analytical use means that a type of protein is taken so that it can be used for research and tests in the laboratory. For example, a certain protein goes through protein purification so that scientists can study its composition, uses, side effects, and quantity needed in order for it to be useful to people. One of the first proteins studied was peptin.
Protein purification goes through several steps. For a simplified version of it, those steps are preparation, extraction, and visualization and assessment.
In preparation, the scientists are preparing to isolate and extract the protein needed for observation. If the protein is excreted by the human body, then this will just be easy. However, there are a lot of other proteins that are not excreted, so a crude extract should be prepared. The crude extract is prepared through centrifugation. The cellular debris is created using cell lysis and the remaining debris is removed by centrifugation.
However, if a thermostable enzyme is needed, a different approach is made. A mixture is taken where the thermostable enzyme is present and it is heated at high temperature. Right after, it is cooled so that the natural characteristics of the enzyme that make it sensitive to temperature dissolves or is recreated to other properties.
The next step is extraction. This was formerly done using precipitation. This is done by letting the nucleic acids in the crude extract turn into water with the help of salt ammonium sulfate. Take note that depending on the nucleic acid you want to extract, the salt and ammonium sulfate concentration differs. Usually, a higher concentration is needed for proteins that have a lower molecular weight.
- Visualization and Assessment
And finally, there’s visualization and assessment. In this step, a lot of chromatography methods can be used depending on the kind of protein. There’s reverse-phase chromatography which is based on separating proteins using hydrophobicities. There is also a size-exclusion chromatography that separates proteins based on the size of the molecules. And then there’s affinity chromatography that is often a final step to the purification process.