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Yeast Protein Expression Process

The expression systems used for recombinant drug production mainly include prokaryotic expression system, mammalian expression system, plant cell expression system, insect cell expression system and yeast animal cell expression system. The yeast expression system is a cost-effective eukaryotic protein expression system that can be successfully expressed intracellularly or secreted, and is suitable for industrial scale-up due to its relatively inexpensive scale-up medium and less demanding culture conditions. Like mammalian cell expression system, yeast protein expression system can modify the expressed protein to ensure the natural conformation of the protein, such as glycosylation, acylation, lipoylation and phosphatization, and can be used to prepare protein raw materials with high added value, which are very close to natural proteins. 

Commonly used yeast expression systems: Saccharomyces cerevisiae expression system, methanol trophic yeast expression system, Schizosaccharomyces expression system. Taking the expression of foreign genes by Pichia pastoris as an example, it includes the following steps:

1. Cloning the target gene into Pichia pastoris expression vector;

2. After obtaining the positive recombinant expression plasmid, cut the positive recombinant plasmid with appropriate restriction endonuclease to linearize it;

3. Transforming the linearized positive recombinant plasmid into a yeast strain (such as GS 115);

4. Inoculate transformants into HIS4 defective plates for the first round of screening, and select G418 plates with different concentrations for the second round of screening;

5. Selecting 10-20 clones for small-scale induction culture;

6. Identify the expression level of foreign genes, select high-level expression strains for large-scale induction culture, so as to prepare the expression protein of foreign genes; 

Main advantages of Pichia pastoris expression system:

1. It has a promoter of alcohol oxidase (AOX) gene, which can be strictly regulated to achieve high-level expression;

2. High-density cell culture can be carried out, and the fermentation tank can reach 120 g/L;

3. It has the function of post-translational modification of eukaryotic cells, and the protein has more biological activity;

4. High level secretion and expression of foreign protein is beneficial to purification;

5. It can realize intracellular expression and extracellular secretion expression; 

At present, there are at least four different methods to introduce foreign plasmid DNA into Pichia pastoris: protoplast transformation, lithium chloride (LiCl) transformation, PEG1000 transformation and electroporation. Among them, electroporation is the most commonly used.