The prokaryotic protein expression system is both the most commonly used expression system and the most economical one. The prokaryotic expression system is represented by Escherichia coli expression system, which has the advantages of clear genetic background, low cost, high expression and relatively simple separation and purification of expressed products. The disadvantages are mainly the lack of protein translation mechanism, such as the formation of disulfide bonds, protein glycosylation and correct folding; thus getting a bioactive protein is less likely.
Yeast protein expression system is represented by Pichia pastoris, which has the advantages of high expression, high induction, high glycosylation mechanism close to higher eukaryotes, easy purification of secreted protein, easy realization of high density fermentation and so on. The disadvantage is that some protein products are easy to degrade and the expression is uncontrollable.
The main advantages of mammalian cell and insect cell expression systems are that the post-translational processing mechanism is closest to the natural form of the body and is most likely to retain biological activity. The disadvantage is that the expression level is usually low, and the stable cell line is difficult to build and the production cost is high.
Protein purification uses the similarity and difference between the different proteins, the use of similarity between the various proteins to remove non-protein material contamination and the use of protein differences in the target protein from other proteins purified. The size, shape, charge, hydrophobicity, solubility and biological activity of each protein are different. These proteins can be extracted from the mixture, such as Escherichia coli lysate, to obtain the recombinant protein.
Protein purification is roughly divided into two stages: the crude separation stage and the fine purification stage. General protein purification is the resin method.
The coarse separation phase mainly separates the target protein from other cellular components such as DNA, RNA and so on. Because of the large volume of the sample and the high volume, high flow rate, large particle size and wide particle size distribution, the protein is separated from the contaminant and, if necessary, a corresponding protective agent (e.g., a protease inhibitor) may be added to prevent degradation of the protein of interest.
Fine purification stage requires a higher resolution. This stage is to the purpose of the protein and that molecular weight and physical and chemical properties of the protein close to the use of smaller resin particles to improve the resolution, commonly used ion exchange column and hydrophobic column, the application should take into account two factors: the resin selectivity and column efficiency.
Selectivity refers to the specificity of the binding of the resin to the protein of interest. The column efficiency refers to the ability of each protein component to elute from the resin one by one. The narrower the elution peak, the better the column efficiency.
With the higher cost of independent research and development of pharmaceutical companies, more and more companies look for outsource companies, but finding a good partner is also important.
Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. Medicilon offers a variety of recombinant protein expression platforms along with a host of other protein services like chemical protein synthesis, protein refolding and structural biology services.
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