The Pichia pastoris expression system is a eukaryotic expression system that has been rapidly developed and widely used in recent years.It is a methanol-nutrient yeast with two ethanol oxidase (Alcohol oxidase, AOX) coding genes, AOX1 and AOX2, both of which have similar sequences, and the AOX1 gene is strictly induced and regulated by methanol. When methanol is the only carbon source, the AOX1 promoter can be induced by methanol to initiate the expression of ethanol oxidase, which can be metabolized with methanol, and plasmids containing the AOX1 promoter can be used to promote the expression of target genes encoding exogenous proteins.

1. Unique and powerful AOX (alcohol oxidase gene) promoter, the expression of exogenous genes can be strictly regulated with methanol.

2. The product is easy to be separated, and the fermentation medium used by Beechcraft yeast is quite cheap. Generally the carbon source is glycerol or glucose and methanol, and the rest is inorganic salt.The medium does not contain protein, which is conducive to the separation and purification of downstream products.

3. Exogenous protein genes are genetically stable, and it can be integrated into the genome of Saccharomyces cerevisiae with high copy number, which is not easy to be lost and can obtain high expression strains.

4. As a eukaryotic expression system, Saccharomyces cerevisiae has the subcellular structure of eukaryotic organisms, and features with post-translational modification such as glycosylation, fatty acylation and protein phosphorylation.

Features for Expression Strain:

GS115: Both AOX1 and AOX2 genes normally express AOX protein (ethanol oxidase), methanol utilization efficiency is high, close to the wild type, and belongs to the rapid methanol utilization type (Mut+)

KM71: AOX1 gene is mostly eliminated, and AOX2 can only be used to encode AOX. The rate of methanol utilization becomes slower, and behaves as a methanol slow utilization type (Muts)

Differences in the integration of exogenous genes lead to different phenotypes in the expressing strains

Table 1. Phenotypes of Yeast Strains

Table 2. Expression Vector Information

For extracellularly expressed proteins, in addition to the signal peptides that can be used with the vector, the following signal peptides are available:

Table 3 . Signal Peptide Sequences

Advantages

1. With a complete Picrorhizobium platform, different experimental programs can be designed according to the protein features or customer needs.

2. High-throughput screening platform, the best expression conditions obtained quickly and effectively

3. Capable of large-scale fermentation and production.

Case 1

Protein A full-length gene was synthesized and subcloned into expression vector pPICZαA, transformed into Saccharomyces cerevisiae strain X33, and protein secretion was successfully expressed (28kDa; C-terminal 6*His) with a yield of 7mg/L and a purity greater than 90%, and the QC graph is as follows:

Protein A Purification QC Table （Reduced SDS-PAGE）

Case 2

Protein B gene was synthesized and subcloned into the expression vector pPIC3.5K for intracellular expression by transforming Saccharomyces cerevisiae strains GS115/KM71, respectively, and the results of SDS-PAGE showed that the protein B (40 kDa after glycosylation) was significantly expressed, among which the clones GS115-5# (24h, 0.5% methanol), GS115-2# (48h, 1% methanol), and GS115-3# (48h, 1% methanol) all successfully expressed the target protein.

 Protein B Expression Test Table （Reduced SDS-PAGE）