Unit 6: Protein Purification
Introduction
The protein purification refers to the processes intended to isolate one or a few proteins from a complex mixture, to a pure solution. It is vital for characterization of the function, structure and interactions of the protein of interest, which allows scientists to better understand the structure and function of the protein. The purification process usually take advantage in the differences of protein size, physico-chemical properties, binding affinity and biological activity.
Please note that some proteins are excreted by the cell while some does not, thus the extraction and purification process is different from the 2. However for the time being, we will only focus on the non-secreted proteins form the cell.
Common methods
The preliminary steps involves various strategies to extract cytoplasmic content of the cell, and then purifying it. One of the common methods is the use of polyhistidine tags with affinity chromatography, which takes advantage of the proteins affinity (the tendency and readiness to bind with other substances).
The use of Tags introduced by recombinant technology is very common in extracting the protein. It is an amino acid motif that consists of at least six histidine (His) residues, which is added into the protein by adding the repetitive histidine codons (CAT or CAC) at either the N-terminal (preceded by Methionine) or the C-terminal (before a stop codon) in the coding sequence of the protein. The gene (consisting the His-tag) will then be transcripted and translated into a protein with a special motif of repeated Histidines. Bacterial cells can be harvested via centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and enzymes such as lysozyme or any combination of these. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound divalent nickel or cobalt ions, where it binds with the chelator with micromolar affinity. By using an affinity chromatographer, it can be purified with high purity. The polyhistidine tag can then be removed endopeptidase.
Implications
The major reason for protein purification is to isolate the desired protein, which can then be used for various biochemical to further understands it, or to use it as some sort of products.
A good example of protein purification is the large scale industrial production of insulin,which the pharmaceutical usage of insulin is mainly to treat diabetic patients. Before when recombinant technology was developed, insulin can only be extracted from cows or pigs, which is a very costly process and repulsion of the insulin is also prevalent. With the help of recombinant technology, human insulin can be produced by bacteria. To do this, an insulin gene is ligated to the plasmid backbone and is transformed into the cell. However, to extract the insulin protein from the cell, protein purification is required. Affinity chromatography is commonly used in extracting the protein by its affinity to the chromatographer.