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In vitro recombinant protein production is a protein obtained by applying recombinant DNA or recombinant RNA technology. Catalog The use of in vitro protein expression technology to extensively produce various types of recombinant proteins, which are used in the fields of medicine, food, daily chemical and other fields. The following briefly introduces the main expression systems of the main recombinant proteins.
Escherichia coli has a clear genetic background, low cost, multiple vectors and hosts to choose from. It is not only the most commonly used expression system, but also the most economical protein expression system. The expression system is optimized, such as the compatibility test of the expression vector and the expression strain. , The optimization of induced expression conditions, renaturation, etc., can greatly solve the difficult problems of protein expression and insoluble protein expression. Suitable for proteins that do not require high glycosylation function.
The genetic background is clear; the reproduction is fast, the cost is low, and the anti-pollution ability is strong; the expression is high, the isolation and purification of the expression product is relatively simple, and the stability is good; the types of commercial vectors and strains are very complete, and the scope of application is wide.
① It does not have the function of eukaryotic post-transcriptional processing and cannot carry out mRNA splicing, so it can only express cDNA but not eukaryotic genomic genes;
② Without the function of eukaryotic post-translational processing, the protein produced by expression cannot be modified by glycosylation, phosphorylation, etc., and it is difficult to form correct disulfide bond pairing and spatial conformation folding.
③ The expressed protein is often insoluble and will aggregate into inclusion bodies in the bacteria, especially when the amount of expressed target protein exceeds 10% of the total bacterial body protein, inclusion bodies are easily formed. The reasons for the formation of inclusion bodies may be that the protein synthesis is too fast, the polypeptide chains are entangled with each other, and the lack of factors to make the polypeptide chains fold correctly, resulting in the exposure of hydrophobic genes. After bacterial lysis, the centrifugal precipitation of the inclusion bodies is conducive to the preliminary purification of the target protein, but the insoluble protein without biological activity must be renatured to re-spread and refold into a natural protein conformation and good condition. Biologically active protein is often a very difficult task. The vector can also be designed to make E. coli secrete and express the soluble target protein, but the expression level is often not high.
④ Some pyrogenic sources (endotoxins) may be produced, and E. coli itself contains endotoxins and toxic proteins. The purification of the target protein needs to remove endotoxins, which also limits its application.
Medicilon is a leading provider of comprehensive, high quality recombinant protein and bioprocess services. We offer 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.
Medicilon researchers established a well-developed multiple protein expression systems and purification services platform, which offers the expression and purification services of a variety of recombinant proteins, with a fast turn-around time and competitive pricing.
The use of Saccharomyces cerevisiae to express recombinant proteins began in 1981. Saccharomyces cerevisiae is suitable for expressing scientifically and commercially valuable heterologous proteins. Because it does not produce toxins, combined with its extensive experience in food production, it has become the best host bacteria for the production of pharmaceutical proteins. A series of insulins produced by Saccharomyces cerevisiae, which are widely used in the treatment of diabetes around the world, is one of the important examples of the application of yeast in the field of biotechnology.
High expression level, a good choice for secreted protein or cell expression, low cost, simple culture conditions, strong scalability, post-translational modifications of most eukaryotes, effective protein folding, and no endotoxin.
The expression level is lower than that of Pichia pastoris, and the secretion capacity may be lower than that of Pichia pastoris. Glycosylation is different from mammalian cells. The structure of over-glycosylated N-terminal glycosyl chain is sensitizing.
Methanol yeast is a yeast that can use methanol as the sole carbon source. There are three main types: H. Polymorpha, Candida Bodinii, and Pichia Pastoris. Among them, Pichia Pastoris (Pichia pastoris) is the most widely used gene expression system. Compared with previous gene expression systems, it has unmatched high expression characteristics and has been regarded as one of the most promising tools for protein production.
High expression level, low cost, simple culture, short production cycle, strong scalability, good choice for secreted protein or intracellular expression, efficient protein secretion and simple purification, extensive post-translational modification, N-terminal glycosylation ability Better than Saccharomyces cerevisiae, no endotoxin.
Using methanol as an inducer has certain hazards, and glycosylation is still different from mammalian cells.
Insect cell baculovirus expression system uses insect baculovirus as the foreign gene carrier, and insects and insect cells as the receptor expression system. Insect cells and mammalian cells have similar translation and post-translational protein modification modes and capabilities, including glycosylation, phosphorylation, acylation, signal peptide excision, and peptide cleavage and decomposition, etc., resulting in recombinant protein antigenicity and immunogen The biological activities such as sex and function are similar to those of natural proteins; it can accommodate inserts of large molecules; it is easy to realize large-scale low-cost production. This system is increasingly being valued and widely used.
High expression level (especially intracellular proteins), faster growth rate, effective cell folding, moderate scalability, extensive post-translational modification, glycosylation is similar to mammalian cells, relatively easy to enzymatically remove Glycosylation (favorable for protein structure determination), no endotoxin.
The culture medium is expensive (the cost of the culture medium has been reduced, and various serum-free medium is widely used), a large amount of virus is required, the secretion pathway of the pro-peptide is inefficient, glycosylation is still different from mammalian cells, and virus infection Can cause cell lysis and potentially degradation of expressed protein.
The expression of foreign recombinant protein in mammalian cells can use plasmid transfection and viral vector infection. Using plasmid transfection to obtain stable transfected cells takes several weeks or even months, while the use of viral expression systems can quickly infect cells and integrate foreign genes into viral vectors within a few days. It is especially suitable for large-scale expression. The target protein was detected in the product. Mammalian cell expression vectors must contain control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminator and polynucleotide signals.
According to the temporal and spatial differences in the expression of the target protein, the expression system can be divided into transient, stable and inducible expression systems. The transient expression system means that the host cell is cultured without selection after the expression vector is introduced. The vector DNA is gradually lost as the cell divides, and the expression time of the target protein is short. The transient expression system has the advantages of simplicity and short experimental period. The stable expression system means that the vector enters the host cell and is selected and cultured, the vector DNA is stably present in the cell, and the expression of the target protein is durable and stable. Due to the need for resistance selection and even pressure amplification and other steps, stable expression is relatively time-consuming and labor-intensive. Induced expression system means that the transcription of the target gene can be opened after being induced by foreign small molecules. The use of heterologous promoters, enhancers and amplifiable genetic markers can increase protein yield.
The host cells commonly used in mammalian cell expression systems include CHO, COS, BHK, SP2/0, NIH/3T3, etc. Different host cells have different effects on protein expression levels and protein glycosylation, so when choosing host cells It should be determined according to the specific situation.
Protein activity: The exogenous protein produced by mammalian cells after translation and processing modification is far better than prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells in terms of activity, and is closer to natural proteins. Mammalian cell expression vectors include prokaryotic sequences, promoters, enhancers, selectable marker genes, terminator and polynucleotide signals.
The biggest advantage of expressing foreign proteins in mammalian cells is the correct high-level structure (such as glycosylation).
The mammalian expression system has unique advantages in protein initiation signal, processing, secretion, and glycosylation, and is suitable for expressing complete macromolecular proteins.
The cost of the culture medium is extremely high, the culture period is long, at least one month or more, the culture is difficult, and the expression level is low.
The composition is complicated, the operation technique is high, the expression output is not large, the yield is low, and sometimes it can cause virus infection.
Animal and plant bioreactors are genetic engineering methods that use common animals or crops as “biological factories” to produce high economic value-added medical proteins, industrial and agricultural enzymes, special carbohydrates, and biochemicals through large-scale cultivation and planting. Degradation of plastics, lipids, and other secondary metabolites and other biological agents.
Animals and plants can express proteins from animals, bacteria, viruses, and plants themselves, which are easy for large-scale cultivation and production, and have special advantages in gene expression and modification and safety. Therefore, the use of animals and plants to produce exogenous proteins has shown extreme Attractive prospects.
In the next period of time, the improvement of biotechnology drugs, new products, new dosage forms, etc. will also be promising. More and more functional proteins with large molecular weight and complex structure will be developed into biotechnology drugs; therapeutic antibodies will become the second innovation climax in the field of biopharmaceuticals; in order to improve drug performance, restructuring, fusion proteinization, and in vitro Chemical modification will receive more and more attention; biosimilars will be standardized and developed on a large scale. With the development of technology, there will be more and more recombinant protein expression systems, and the technology will become more and more mature.
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