Yeast expression system is the most economical and efficient eukaryotic protein expression system, which can successfully achieve intracellular expression or secretory expression, and its amplification medium is relatively cheap, and the culture conditions are not demanding, so it is suitable for industrial amplification. Like mammalian cell expression systems, yeast protein expression systems can perform modifications such as glycosylation, acylation, lipidation, phosphorylation, etc. to ensure the natural conformation of the expressed protein, and can be used to prepare proteins that are very close to natural proteins. High value-added protein materials.
Yeast is a common host for the production of proteins from recombinant DNA. They provide relatively easy genetic manipulation and grow quickly to high cell densities on cheap media. As eukaryotes, they are capable of protein modification, such as glycosylation commonly found in eukaryotic cells, but relatively rare in bacteria. Therefore, yeast can produce complex proteins that are the same or very similar to natural products from plants or mammals.
The first yeast expression platform was based on the baker’s yeast Saccharomyces cerevisiae. However, various yeast expression systems have been studied and are widely used in various applications based on their different characteristics and capabilities. For example, some of them grow on a wide range of carbon source bases and are not limited to glucose, just like baker’s yeast. Some of them are also used in genetic engineering and the production of foreign proteins. The various yeast expression platforms differ in several characteristics, including their productivity and the ability to secrete, process, and modify proteins in specific instances. However, the use of all expression platforms has some basic similarities.
The yeast expression system has been used to produce expression products of foreign genes for more than ten years. Among them, the S. cerevisiae expression system and the P. pastoris expression system are the most popular. Up to now, people have successfully expressed many proteins of a variety of organisms (including bacteria, fungi, viruses, protists, plants, vertebrates, and humans) using yeast expression systems, including enzymes, proteasomes, proteasome inhibitors, Receptors, single-chain antibodies, antigens, regulatory proteins and other proteins.
In the medical field, the yeast expression system has been successfully applied to the preparation of genetic engineering vaccines, the preparation of genetic engineering drugs (such as antibody drugs, protein drugs), and protein function research. Abroad, insulin-like growth factor 1 (IGF-1) and human serum albumin (HSA), which are used in the treatment of amyotrophic lateral sclerosis, have passed clinical trials and are waiting for FDA approval before they can enter clinical use.
The CoMed basic vector contains all E. coli elements for propagation in the E. coli system and MCS (polyclonal ste) for integration of ARS, rDNA, selection marker and expression cassette modules. The ARS fragment is flanked by the restriction sites of Sac II and Bcu I, the rDNA region of the restriction sites of Bcu I and Eco 47III, the selection markers of the restriction sites of Eco 47III and SalI, and the restriction sites of Sal I and Apa I Promoter element. In order to produce the desired product, a suitable yeast strain is transformed with a vector that contains all the necessary genetic elements for the production of the biological product of interest.
The vector must also contain a selection marker, and the selection marker needs to select yeast from the vectors that have been successfully ingested. The vector also contains certain DNA elements that allow the yeast to incorporate foreign DNA into the yeast’s chromosome and replicate it. Most importantly, the vector contains a segment responsible for the production of the desired compound, called an expression cassette. The expression cassette contains a series of regulatory elements that are used to control the degree and degree of final manufacturing of a certain product. Next comes the genes of the biological product itself. The expression cassette is terminated by a terminator sequence, which stops the transcription of the expressed gene.
Saccharomyces cerevisiae expression system, methanolotrophic yeast expression system, Schizogenesis pombe expression system. Taking the expression of foreign genes in Pichia pastoris as an example, it includes the following steps:
Clone the target gene into the Pichia pastoris expression vector, harvest the positive recombinant expression plasmid and digest the positive recombinant plasmid with appropriate restriction enzymes to linearize it. Transform the linearized positive recombinant plasmid into the Pichia pastoris strain ( For example, GS115, KM 71) Inoculate the transformant on HIS4-deficient plates for the first round of screening. Use G418 plates with different concentrations for the second round of screening. Select 10-20 clones for small-scale induction culture, identify the expression of foreign genes, and select High-level expression strains undergo large-scale induction culture to prepare foreign gene expression proteins.
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