Protein purification is a procedure that involves a chain of methods aimed at isolating either one or more proteins from whole organisms, tissues, cells, or complex mixtures. It plays a huge role in the characterisation of interactions, structure, and function. This process separates protein from non-protein parts of a complex mixture. It does also help separate the desired protein from all other proteins.
The truth is that separating a single protein from other types of proteins is the most involved process in protein purification. This process generally exploits different protein size, binding affinity, physicochemical properties, and biological activity. The pure result of this process is called protein isolate. Fleet bioprocessing explore and study the concepts and compounds of protein purification.
The Purpose of Protein Purification
Protein purification can either be analytical or preparative. What does this mean? Well:
Analytical Purification Procedure
This is a procedure that involves collecting a relatively small amount of protein simply for various types of research or analysis. This may include conducting a study, quantification, and identification of the protein structure, function, and post-translational modifications.
Preparative Purification Process
This is a process that involves collecting relatively large quantities of purified protein for various successive uses. For example, the preparative procedure may be used for commercial purposes like biopharmaceuticals (example, production of insulin), nutritional protein (example, the production of soy protein isolate), and enzymes (example, production of lactase). Some preparative purification processes involve complete removal of bi-products such as host cell proteins, which may be a potential threat to a patient’s health.
Protein Purification Strategies
The key to designing a purification process is based on the choice of the starting material. In both animals and plants, the particular protein usually is not distributed homogeneously through the body. That is because different tissues or organisms have different values of proteins. In other words, different tissues or organs will either have a high or low concentration of protein. For this reason, protein purification is only used in organs or tissues with the highest concentration of protein to produce a given amount of purified protein.
If the protein is available in low concentration, then scientists may move on to DNA technology to develop cells that produce large quantities of the desired protein. This technology is known as an expression system. It is a procedure that allows the protein to be targeted; through the process such as Strep-tag or His-tag to facilitate purification. This reduces the number of purification steps needed.
Analytical purification process generally uses 3 properties to detach protein. The first protein may be purified based on their isoelectric point. This is done by running the material through an ion-exchange column or pH graded gel. The second protein may be separated based on molecular size or weight using a size exclusion chromatography or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) processes.
2D-PAGE is often used to purify protein which is then analysed using peptide mass fingerprinting to identify its identity. This is important scientifically because it helps determine the limit of the protein. The third protein may be separated by hydrophobicity or polarity using reversed-phase chromatography or high performing liquid chromatography.
The truth is that protein purification procedure comprises one or more chromatographic stages. That is because different protein interacts different with the column material and therefore can be separated by the number of times it is passed through the column.