Protein purification is essential in characterising the interactions, function, and structure of a specific protein. It is a series of processes that isolate one or several proteins from a whole organism, tissues, cells, or complex mixture, separating the protein from non-protein parts in a sample and then identifying and separating a target protein type from other proteins.
Separating a specific protein, such as an ADA antibody, from all others is the most demanding aspect of the purification process. The steps covered exploit various targeted protein attributes, such as binding affinity, size, biological activity, and physico-chemical properties. The results, if pure, are called protein isolate.
What is its Purpose?
Protein purification can be analytical or preparative.
• Analytical protein purification results in small protein amounts for various analytical or research purposes like quantification, protein structure studies, protein function, identification, and post-translation modifications. Urease and pepsin were the first proteins to be purified to the degree of being crystallised.
• Preparative protein purification products a more considerable amount of purified proteins for purposes like preparation of commercial products ranging from biopharmaceuticals like insulin, enzymes such as lactase, and healthy proteins like soy protein isolate. Preparative purification will involve several steps in the removal of bi-product. An excellent example is the extraction of host cell proteins that can be a potential health risk to patients.
The starting material choice is crucial to the purification process’s design. Animals and plants have a protein that is not homogeneously distributed across the entire body. Different tissues or organs have lower or higher concentrations of the protein. The use of tissues or organs with the highest concentration of the protein reduces the volume required to produce a specified purified protein amount.
If the sample has a low concentration or a high value, recombinant DNA technology could develop cells that produce large quantities of the protein of interest. It is a process called Expression. Recombinant Expression allows for better protein targeting, like by a Strep-tag or His-tag, to facilitate the purification process therein, reducing the steps involved.
Analytical protein purification utilised three elements in protein separation.
1) The first is the purification of the proteins according to their isoelectric points. It is a process that entails running them through an ion-exchange column or pH-graded gel.
2) The next step is separating the protein depending on their molecular weight and size, which is done via Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS -PAGE) analysis or Size Exclusion Chromatography.
Proteins are often purified using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes, and the detection limits for protein are nowadays meagre, and nanogram quantities of the protein that are enough for examination.
3) Third and last, the protein could be parted via reserved-phase chromatography or hydrophobicity/polarity through high-performance liquid chromatography.
Protein purification protocol typically follows one or more chromatographic stages. The basic one is to have the protein-laced solution flowing through a column packed with different materials. Various proteins interact differently with the materials and are separated by the time they pass through the column from one end to the next.
Conversely, this can be perpetuated by the essential conditions needed for eluting the protein from the column. Protein detection often manifests as the proteins come off the column based on their absorbance at 280mn.