Protein purification is a process of isolating one or a few types of proteins from a complex mixture found in cells, tissues or organisms. It is used for characterisation of the function, interactions and structure of the specific type of protein in question. The purification process separates the protein and the non-protein parts from the overall mixture.
It is also used to separate the specific protein from all the other types of proteins. Separation one type of protein from the rest is the difficult part of the protein purification process. The separation process exploits the differences in the size of the protein, binding affinity, physico-chemical properties and biological activity. The resulting protein is referred to as protein isolate.
Protein purification is either analytical or preparative, and often links to an ADA response. The latter produces a large quantity of purified proteins for subsequent use. Some examples include the preparation of commercial products such as enzymes (lactase), nutritional proteins (soy protein isolate) and biopharmaceuticals (insulin). There are several preparative purifications steps often used to remove bi-products such as host cell proteins. They often pose a potential threat to the health of the patient.
Analytical purification, on the other hand, produces a smaller amount of protein often used for research and analytical purposes. These include identification, studies of the structure of the protein, quantification, function and post translational modifications. Urease and pepsin were the first proteins to undergo the purification process such that they could be crystallised.
Stages of Purification
The choice of a starting material holds the key to the entire purification process. Normally, a particular protein isn’t usually distributed homogeneously in the body of plants or animals. Different tissues and organs have varying concentration of the specific protein. Using the organs or tissues with the highest concentration of the protein reduces the volumes required to produce a specific amount of purified protein.
If the protein concentrations are available in low volumes or if it has a very high value, researchers use recombinant DNA technology to develop cells that produce large quantities of the desired protein (referred to as expression system). Here, the protein will be tagged to reduce the overall steps required in the purification process.
In analytical protein purification, researchers utilise 3 properties to separate the proteins. First, the proteins will be purified depending on their isoelectric points where they are run through an ion exchange column or a PH graded gel. Next, the proteins will be separated depending on their size and overall molecular weight through SDS PAGE or size exclusion chromatography.
Proteins will be purified using 2D-PAGE then analysed through fingerprinting of the peptide mass to identify the most important protein identify. It’s a useful procedure in the scientific world since the detection limits for proteins are very low currently. With this procedure, only nanograms of proteins are used for the analysis process. Lastly, proteins are separated by hydrophobicity/polarity through reversed-phase or high-performance liquid chromatography.
A simple protein purification process involves one or a few chromatographic steps. Here, the procedure involves flowing the solution of the protein through a column filled with various materials. Different types of proteins will react differently in the material and will be separated by the time they pass the column or the conditions required to elute the protein from the column. The proteins will be detected as they are coming through the column because of their absorbance rate at 280mm.