Protein purification is the expression of recombinant proteins through using bacterial vectors and hosts in a mature technology. In carrying out biological research, recombinant protein purification is essential. The recombinant protein must be isolated and purified from the organism. The method involved in protein purification hinges on the similarity and differences between different recombinant proteins.
Based on the similarity between proteins, non-proteinaceous can be removed. Then, the recombinant target protein can be isolated and purified through the differences between proteins for the protein expression company. The protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches.
Expression of recombinant proteins
In the expression of recombinant proteins, when bacterial vectors and host are used, recombinant proteins are always in a matured technology. And this method is with the appropriate cDNA and PCR for the rapid production of expression plasmids. These plasmids are transformed into expression hosts following the constructs of sequence determination, and single colonies are picked and fermentation performed.
To generate ̴ 50 to 80g wet weight of cells, E. coli with a 2 liters formation complex media will be used. If there is a modest protein expression between 100 to 300 mg of recombinant protein in the cells, the problem will be how to isolate it in its active form. If it is a soluble protein, it can be recovered with good yields of less than 50%. In comparison, insoluble proteins that need to undergo denaturation and the folding cycle can be recovered with more modest yields of 5% to 20%. Therefore, using small-scale fermentations and laboratory-scale processing equipment, proteins can be produced through protein production cro in more quantities of about 10 to 100mg.
Chromatography for purification
The method of purification of recombinant proteins through chromatography is very popular and involves a set of different techniques. In the use of chromatography techniques, there is separation based on differences between the properties of the protein to be purified and the properties of other molecules of the sample. The properties can vary from specific structures of the molecule, the electric charge, or the size of the molecule, among many other things. In protein purification, the method that is mostly used is affinity chromatography. This method separates proteins based on their specific interaction with a matrix.
This technique is one of the most effective because it takes advantage of incorporating a structure of choice, which is tagged onto the protein. This tag that is available in this protein separation technique is not present in any other sample molecule. This conferred on the target protein the specific characteristics that will be used in distinguishing and separating from the rest.
In some other instances, when carrying out custom recombinant protein production purification, a tag cannot be added to the molecule. In this situation, less specific methods will be used, and they can also be equally effective. One of these methods is gel filtration which separates molecules according to their size. Another method commonly used is ion exchange chromatography, which separates molecules according to the electric charge in certain pH and temperature conditions. Hydrophobic interaction chromatography and reversed phase chromatography are other methods that separate proteins based on polarity.
What makes the difference between these two methods is the matrix polarity through which proteins to be purified interact. Hydrophobic interaction chromatography and reversed phase chromatography are less specific than affinity chromatography as they won’t separate well. Therefore, using any of these techniques they are usually performed in combinations of two or more to achieve good results. The two methods work well with affinity chromatography to achieve more refined results.
The principle of protein purification
In protein production service, different recombinant proteins have different amino acid sequences and spatial structures that result in differences in their properties from biological, physical, and chemical. Based on the differences in the properties of the desired protein to be separated, a reasonable protein purification strategy can be designed from other proteins and lysates.
To achieve the level of purity that is aimed at, most purification protocols require more than a single procedure. It is best practice to have a few purification steps that can be gotten because each step in the process will bring about some loss of product with an assumed yield of 80%.
Having background information on assays and sample procedures in place helps consider the three-phase purification strategies. The purification has three phases: the capture phase, intermediate, and polishing with each specific objective. In the capture phase, the objectives are to isolate, concentrate, and stabilize the target procedure.
The selection and combination of purification techniques are essential in applying purification principles. This combination aims to evolve a route that will be the fastest to a product of required purity. And with any chromatographic separation, each technique will offer a different performance.