CHO cell expression system and Pichia pastoris expression system are two expression systems with promising development prospects. In order to have a more intuitive understanding of the two expression systems, we deliberately make a certain comparison between the two expression systems in this article So that you can have a deeper understanding of the two expression systems
1. CHO cell expression system (1) Advantages CHO cells are fibroblasts and can grow adherently. It can also be grown in suspension. Currently used CHO cells include original CHO and dihydrofolate reductase diploid gene deletion type (DHFR-) mutant strain CHO. In recent years, in order to reduce production costs and reduce the potential harm caused by blood products, animal cell production began to use serum-free medium (SFM), but SFM often results in poor cell viability, poor adhesion, and poor ability to secrete foreign proteins And other disadvantages. Another researcher tried to transfer the insulin-like growth factor IGF gene and transferrin gene into CHO cells to obtain "super CHO" that can secrete the necessary proteins by itself. Without transferring transferrin and insulin in the medium, the cells can grow well in SFM . Compared with other expression systems, CHO expression system has the following advantages:
(1) With accurate post-transcriptional modification function, the expressed protein is closest to the natural protein molecule in terms of molecular structure, physical and chemical properties and biological function;
(2) It can be adhered to the wall and can also be cultured in suspension, and has a high ability to withstand shear force and osmotic pressure;
(3) It has the ability of efficient amplification and expression of recombinant genes, and the integration of foreign proteins is stable;
(4) It has the function of extracellular secretion of products, and rarely secretes its own endogenous proteins, which facilitates the separation and purification of downstream products;
(5) High-density culture can be achieved by suspension culture or in serum-free medium. And the culture volume can reach more than 1000L, which can be mass-produced.
(2) Existing problems In the past few decades, humans have conducted a lot of research and development on animal cell culture technology, and have made great progress, but the technical level of using CHO cells to express foreign genes has not yet met biological drugs. The current development and production requirements, the current main problems in the upstream work are as follows:
â‘ The constructed recombinant CHO cells have low production efficiency and low product concentration;
â‘¡Some glycosylated expression products are unstable and difficult to purify;
â‘¢ The upstream construction of the recombinant CHO cells is disconnected from the downstream separation and purification, which mainly shows that the upstream construction focuses on its high-efficiency expression, and whether the products expressed by the higher education can be effectively extracted, that is, the separation and purification process is less considered;
2. Pichia pastoris cell expression system (1) Features Since Gregg et al. First expressed hepatitis B surface antigen (HBsAg) in Pichia pastoris to 1995, there have been more than forty foreign proteins in Pichia pastoris host bacteria Get expression. In recent years, there have been dozens of exogenous genes reported in Pichia pastoris, and more than one year from year to year. Compared with other expression systems, the Pichia pastoris expression system has the following advantages:
1) It contains a unique and powerful AOX (alcohol oxidase gene) promoter, which can strictly regulate the expression of foreign genes with methanol;
2) The expression level is high, it can be expressed in the cell, and it can also be secreted. In Pichia pastoris, the highest reported amount of tetanus toxin C is 12g / l, generally greater than 1g / l. The vast majority of foreign genes are expressed at higher levels than in bacteria, Saccharomyces cerevisiae, and animal cells. Generally, the foreign gene in Pichia pastoris carries a signal peptide sequence to guide secretion, so that the expressed foreign target protein is secreted into the fermentation broth, which is beneficial to separation and purification;
3) The fermentation process is mature and easy to enlarge. There is already a large-scale industrial high-density fermentation process, and the cell dry weight is more than 100g / l. When expressing recombinant protein, it has been successfully scaled up to 10,000 liters;
4) Low cultivation cost and easy separation of products. The fermentation medium used by Pichia pastoris is very cheap, the general carbon source is glycerol or glucose and methanol, and the rest is inorganic salts. The medium contains no protein, which is conducive to the separation and purification of downstream products. Of galactose;
5) The genetic stability of foreign protein genes is stable. Generally, the foreign protein gene is integrated into the chromosome of Pichia pastoris, and it replicates with the chromosome replication, and is not easy to be lost;
6) As a eukaryotic expression system, Pichia pastoris has a subcellular structure of eukaryotes, and has post-translational modification processing functions such as glycosylation, fatty acylation, and protein phosphorylation.
(2) Existing problems Although the methanol nutritional yeast expression system is considered to be a promising eukaryotic expression system, many expression products have been used in clinical treatment and prevention, but it is not a versatile expression system, as a new The expression system, like other expression systems, is not as satisfactory as the Pap system. It also has its limitations, and there are still some defects or deficiencies. Such as the heterogeneity of secreted products, including the presence of polymers, the fermentation period is long, easy to contaminate, and long-term fermentation is not conducive to the expression of foreign proteins; the use of flammable methanol as a raw material requires hygienic safety of the expressed product Consider; the high cost of screening drugs also brings limitations to production applications; examples of low or no expression are also increasing. The incomplete processing of signal peptide, imperfect modification function and internal degradation have brought difficulties to the purification and industrialization of foreign proteins. Moreover, compared with the safe and reliable S. cerevisiae with clear background, the Pasteur system has other disadvantages:
(1) The research foundation of molecular biology is poor, and it is more difficult to genetically modify it.
(2) It is not a food microorganism, and methanol is added during fermentation. Therefore, it has not been widely accepted to use it to produce drugs or food.
(3) Although fermentation can reach a high density, the fermentation cycle is generally longer.
One of the most important defects is that secreted proteins are degraded by self-secreted proteases in the medium. Since methanol yeast uses high-density fermentation, proteases will also increase with the increase in cell density.
The following three methods can effectively reduce the degradation of foreign proteins during fermentation: â‘ Add amino acid-rich additives, such as tryptone, which can be used as protease substrates to be decomposed to reduce the degradation of the target protein; â‘¡Change the medium pH value, because Pichia pastoris can grow normally in the range of pH value 3.0 ~ 7.0, adjusting the pH value can change the activity of protease, thereby reducing protein degradation; â‘¢ application of protease-deficient strains, such as: SMD1163, 1165, 1168 .
Second, some proteins are expressed very low or not expressed in Pichia pastoris. The reason has not been fully elucidated so far, but may be related to the following aspects:
â‘ Pichia pastoris, like humans and other species, has different preferences for the amino acid codons used, which may limit its protein translation speed. Comparing the usage of 110 yeast gene codons, it was found that all genes can be divided into two distinct groups: high expression group and low expression group. The tRNA corresponding to the codons of the genes in the high expression group was also significantly higher than that in the low expression group, the proportion of the third codon as cytosine was also significantly increased, and the AT content was relatively low. Identification of the least frequently used codons in yeast and humans found that 6 out of 8 low-frequency codons are the same. No matter E. coli, Drosophila, or yeast and humans, the low-frequency codons in a large number of expressed protein genes are significantly reduced. The large amount of low-frequency codon content protein expression is harmful to itself. In order to express the HIV-2 coat glycoprotein gpl05 in Pichia pastoris, Zhang YJ et al. Mutated the low-frequency codon AGG to the homologous CGA, and the GAT encoding aspartic acid to GAA encoding glutamic acid, and introduced yeast The preferred TCC finally achieved high expression of gpl05 in it;
â‘¡When the integrated copy number increases, it may produce epigenetic regulation at the transcription level, affecting the increase of recombinant protein yield;
â‘¢ Transcription is terminated prematurely, which is mainly caused by excessive AT content. According to reports of HIV. 1 gpl20 cannot be expressed in Pichia pastoris due to the early termination of transcription in AT-rich regions. There are some consensus sequences in Saccharomyces cerevisiae, similar to the AT-rich regions in HIV-1 gpl20 that cause early termination of transcription, such as: After changing these sequences, you can find longer mRNA appears.
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