Size-Exclusion Chromatography in Purification of Proteins Literature Review
Size-exclusion chromatography is a biochemistry process used for a number of uses in the laboratory. The process is used to purify protein, for desalting, buffer exchange, or for separating a group whereby the sample has two major groups. Size-exclusion chromatography is a simple method for separating molecules which have different molecular seizes under mild conditions. It makes use of the size of molecules in solutions to determine the method and means of separation. For instance, spinColumns have short media packaging so that the samples in the solution are separated using the size. Size-exclusion chromatography, also referred as Gel Filtration, is not only used to remove contaminants based on their molecular weight, they are also used for buffer exchange before and after a different chromatography technique. It is also used to remove reagents in order to end a given reaction (Mori, 2013). In Gel Filtration, the maximum decree can be attained with samples that have a volume of 0.5% to 2% of the column volume, although values up to 5% can give an acceptable separation. Larger sample volumes can also be used on condition that the resolution between the target proteins and the impurities to be removed is high. In order to raise the capacity, the sample to be used can be concentrated before the Gel Filtration or else a larger column may be used. The choice of a buffer solution to be used does not affect the resolution. However, a low concentration of salt should be used in order to reduce the weak electrostatic relations between the size-exclusive chromatography media and protein. The choice of the buffer conditions to be used should be the ones that do not cause precipitation or inactivation (Mori, 2013). The conditions should maintain the biomolecules stability and also target the protein activities.
Protein purification is a sequence of procedures which are used to separate a number of proteins from a composite blend. In most case, the mixture is composed of tissues, cells, and whole organisms. The process of protein purification is important when one wants to characterize the functions, the structure, and the interaction of a particular type of protein. The process of purification may first, separate proteins from the non-proteins. After separation of proteins from the non-proteins, the next step is separating the preferred proteins from the other proteins components. Separating proteins from other components of proteins is most difficult process in the purification procedure. The separation process exploits the differences in size of proteins, the binding affinity of the proteins, the physico-chemical properties, and the biological activities of the proteins. After the procedures when the preferred protein has been isolated, the resultant product is referred as a protein isolate. There are several methods that can be used for protein purification. They can be divided into two: preparative methods and analytical methods. The Size-exclusion chromatography falls within the analytical methods. The difference between the two methods is the amount of protein that can be purified within a single experiment (Batas, 1999). The chromatography technique detects and identifies the different proteins in a solution. The preparative methods on the other hand, produce large quantities of the proteins to be used for other purposes. Protein purification can either be analytical or preparative. Preparative purifications takes place when one wishes to produce bulky quantities of purified proteins for other uses. An example is the preparation of enzymes such as lactase, biopharmaceuticals, and nutritional proteins. Analytical purification involves the process of producing small quantities of pure proteins for the purpose of research and analytical purposes (Batas, 1999). The analytical purposes include quantification, identification, and studying the structures of the proteins.
The Size-exclusion chromatography is used for protein purification in solutions or denaturing conditions using the porous gels. The principle used in the technique is that of little molecules having to cross a bigger volume using a permeable medium. Proteins that have different ranges in size will require different volumes of a solvent before it is taken at the end of the pipe of the gel. In this situation of protein purification, the solvent in use is normally united in diverse test tubes. The test tubes that do not contain any measurable trace of protein are usually redundant. The solution that remains is composed of the proteins that need to be purified together with any other sized-proteins. The advantage of using Gel-Filtration is because of the good separation of big molecules from the mixture with a small volume of solvent (Janson, 2012). Also, various solutions can be used without the interference with the filtration process. The technique also ensures that it preserves the biological activity of the molecules to be separated. Size-exclusion chromatography is combined with other techniques that help in the purification of the proteins. During purification of proteins, there are short and well-defined purification time and narrow bands, which results in a higher sensitivity. Further, during purification by Gel-Filtration, there is no sample loss since the solute does not interact with stationary phase of the column. There are demerits of using the method Gel-Filtration for protein purification. One, the number of bands to be accommodated is limited because the time scale of the chromatogram is shorter. Also, there has to be a 10% difference in the mass of the molecules in order to expect quality resolution. Size-exclusion chromatography is the mostly preferred technique of purification because of its routine and validated analysis because of its speed and reproducibility (Janson, 2012).
References
Mori, S., & Barth, H. G. (2013), Size exclusion chromatography, Springer Science & Business Media.
Batas, B., Schiraldi, C., & Chaudhuri, J. B. (1999), Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography, Journal of biotechnology, 68(2), 149-158.
Janson, J. C. (Ed.). (2012), Protein purification: principles, high resolution methods, and applications (Vol. 151), John Wiley & Sons.
