With the development of molecular biology, more and more scientific researchers have mastered various experimental techniques of molecular biology, and developed a complete set of kits to make gene cloning and expression easier and easier. However, the upstream work of molecular biology is often not the ultimate goal. The key to molecular cloning and expression is to obtain pure expression products to study their biological effects, or to produce large quantities of biological products that can be used for disease treatment. Compared with upstream work, the downstream work of molecular cloning is more difficult, and protein purification is very complicated. In addition to ensuring purity, protein products must also maintain their biological activity. The purification process must be able to produce the same quantity and quality of protein every time, with good repeatability. This requires the application of very adaptable methods rather than the best method to obtain pure protein to purify the protein. A good method under laboratory conditions may fail in large-scale production applications, because the latter requires scale and good repeatability in daily applications.
General principles of protein purification
Protein purification must use the inherent similarities and differences between different proteins, use the similarities between various proteins to remove the pollution of non-protein substances, and use the differences of each protein to purify the target protein from other proteins. Each protein has differences in size, shape, charge, hydrophobicity, solubility, and biological activity. Using these differences, the protein can be extracted from a mixture such as E. coli lysate to obtain a recombinant protein. Protein purification can be roughly divided into two stages: coarse separation stage and fine purification stage. The coarse separation stage mainly separates the target protein from other cellular components such as DNA and RNA. Due to the large sample size and mixed components, the resin used requires high capacity, high flow rate, large particles, and wide particle size distribution. And can
In order to quickly separate the protein from the contaminants, to prevent the target protein from being degraded. The fine purification stage requires higher resolution. In this stage, the target protein must be distinguished from those with similar size and physical and chemical properties. Smaller resin particles are used to improve the resolution of commonly used ion exchange columns and hydrophobic columns. Two factors, the selectivity of the resin and the efficiency of the column, should be considered comprehensively when applying. Selectivity refers to the specificity of the binding between the resin and the target protein, and the column efficiency refers to the ability of each component of the protein to be eluted from the resin one by one. The narrower the elution peak, the better the column efficiency. With only good selectivity, the elution peak is too wide, and the protein cannot be separated effectively.