The expression systems used for recombinant drug production mainly include prokaryotic expression systems, mammalian expression systems, plant cell expression systems, insect cell expression systems, and yeast animal cell expression systems. The yeast expression system is a cost-effective eukaryotic protein expression system capable of achieving successful intracellular or secretory expression. Its scale-up medium is relatively inexpensive, and its cultivation conditions are undemanding, making it suitable for industrial scale-up. Like mammalian cell expression systems, the yeast protein expression system can perform post-translational modifications such as glycosylation, acylation, lipidation, and phosphorylation to preserve the natural conformation of proteins. This makes it suitable for producing high-value protein materials that closely resemble natural proteins.

Commonly used yeast expression systems include: Saccharomyces cerevisiae expression systems, methylotrophic yeast expression systems, and Schizosaccharomyces pombe expression systems. Taking Pichia pastoris as an example for foreign gene expression, the process involves the following steps：

1.Clone the target gene into a Pichia pastoris expression vector.

2.After harvesting the positive recombinant expression plasmid, digest it with appropriate restriction enzymes to linearize the recombinant plasmid.

3.Transform the linearized positive recombinant plasmid into Pichia pastoris strains (e.g., GS115).

4.Plate the transformants on HIS4-deficient medium for primary screening, then use G418 plates with different concentrations for secondary screening.

5.Select 10-20 clones for small-scale induction culture.

6.Evaluate the expression level of the foreign gene, and select high-expression strains for large-scale induction culture to produce the target recombinant protein.

The Pichia pastoris expression system offers the following key advantages:

1.Utilizes the AOX1 promoter - The alcohol oxidase (AOX) gene promoter enables strict regulation and achieves high-level expression.

2.Strong aerobic growth preference - Supports high-cell-density cultivation, with fermentation densities reaching up to 120 g/L in bioreactors.

3.Eukaryotic post-translational modification capability - Produces proteins with higher biological activity.

4.High-level secretory expression of foreign proteins - Facilitates downstream purification.

5.Supports both intracellular and extracellular secretory expression - Offers flexibility in protein production strategies.

Currently, there are at least four different methods for introducing foreign plasmid DNA into Pichia pastoris: protoplast transformation, lithium chloride (LiCl) transformation, PEG1000 transformation, and electroporation. Among these, electroporation is the most commonly used.