Advantages and disadvantages, production process, commonly used carriers, multiple copies, and purification methods of Pasteur Pichia pastoris
Pasteur Pichia pastoris is a type of methanol tolerant yeast that can utilize methanol as its sole carbon and energy source.
Like other yeasts, it mainly exists in haploid form during the asexual growth period. When the environment and nutrition are limited, it often induces two different physiological types of haploid cells to mate and fuse into a diploid form.
Another biological characteristic of Pasteur Pichia pastoris is that the alcohol oxidase required for methanol metabolism is sorted into peroxisomes, forming regionalization.
When glucose is used as the carbon source, there is only one or a few small peroxisomes in the bacterial cells, while when methanol is used as the carbon source, peroxisomes account for almost 80% of the entire cell volume, and AOX increases to 35% -40% of the total cell protein.
Therefore, when inserting exogenous protein genes through homologous recombination before the AOX gene, a large amount of expression can be obtained.
At the same time, based on the characteristic of methanol yeast that can form peroxisomes, this system can be used to express some toxic proteins and easily degradable enzymes, as well as to study cell specific regional biogenesis and its mechanisms and functions, providing inspiration for similar research in higher animals.
Koichi Ogata et al. first discovered in 1969 that certain yeasts can grow using methanol as a carbon and energy source (Ogata, et al. 1.1969). Since then, the potential of using methanol utilizing yeasts to produce single-cell proteins as animal feed has attracted widespread attention.
In 1987, Cregg et al. first reported the expression of hepatitis B surface antigen (HbsAg) using methanol fed yeast, and subsequently began collaborative development of the Pichia pastoris expression system. In 1993, Philip Petroleum sold the patent for the Pichia pastoris expression system to Research Corporation Technologies and commissioned Invitrogea to sell the product.
Advantages and disadvantages: Pichia astatoris yeast does not have natural plasmids in its body, so the expression vector needs to undergo homologous recombination with the host chromosome to integrate the exogenous gene expression framework into the chromosome for the expression of exogenous genes.
Including promoters, exogenous gene cloning sites, termination sequences, screening markers, etc. The expression vectors are shuttle plasmids, which are first replicated and amplified in Escherichia coli, and then introduced into host yeast cells. To induce extracellular secretion of the product, the expression vector also needs to carry a signal peptide sequence.
The advantage of type A is that it has the promoter of alcohol oxidase AOX1 gene, which is currently one of the strongest and most strictly regulated promoters;
(2) The expression efficiency is high, and the exogenous proteins expressed can account for more than 90% of the total expressed proteins, which is beneficial for the separation and purification of the target protein;
(3) High density cultivation can be achieved in simple synthetic media;
(4) The expression plasmid can stably integrate in the form of single or multiple copies at specific sites in the genome;
(5) Due to the fact that this yeast can use methanol as a carbon source and energy source, while the vast majority of microorganisms cannot use methanol as a carbon source, it can reduce pollution.
Insufficient A (1) fermentation cycle; (2) Methanol is flammable, explosive, and toxic, posing a certain level of danger; (3) The drugs required for screening high-yield strains are relatively expensive; (4) The culture medium and conditions are not mature.
Production process (1) Cell proliferation and reproduction; (2) Transition stage of batch feeding (glycerol or glucose); (3) Induction expression stage (methanol); The carbon source at each stage is a limiting matrix, and the dynamic model of its replenishment rate is the basis for efficient expression.
The typical expression vector of Pasteur Pichia pastoris contains the promoter and transcription stop codons (5'AOX1 and 3'AOX1) of the alcohol oxidase 1 (AOX1) gene, which are separated by multiple cloning sites (MCS) where exogenous genes can be inserted.
This vector also contains the histone dehydrogenase gene (HIS4) selection marker and the 3'AOX1 region. When the integrated vector transforms the receptor, its 5'AOX1 and 3'AOX1 can recombine with homologous genes on the chromosome, allowing the entire vector to be inserted into the receptor chromosome along with exogenous genes, which are expressed under the control of the 5'AOX1 promoter.
Pichia pastoris itself does not secrete endogenous proteins, while the secretion of exogenous proteins requires signal sequences that guide secretion.
The commonly used host strains of Pichia pastoris include GS115 and KM71, both of which have HIS4 nutritional deficiency markers. Among them, the GS115 strain has the AOX1 gene, which is Mut+, meaning the normal type for methanol utilization;
The AOX1 site of the KM71 strain is inserted with the ARG4 gene, and the phenotype is Muts, which is a slow methanol utilization type. Both strains are suitable for general yeast transformation methods.
Unlike plasmid expression vectors with multiple copies and replication, the copy number of integrated expression vectors can vary greatly. The expression strain containing multiple copies of exogenous genes also synthesizes a higher amount of protein.
In vivo integration can be screened for possible multi copy insertions through high genetic resistance to streptomycin; And in vitro integration can generate tandem insertion of exogenous genes through connections.
There are two ways to obtain multi copy expression strains: one is to use SDS-PAGE electrophoresis, immune hybridization, or bacterial drop hybridization methods for natural screening among a large number of transformants. Obtain high-yield expression strains.
Another approach is to insert multiple expression cassette copies into a single vector before transformation, and then integrate them onto the receptor chromosome through exchange.
Purification method Proteins expressed in yeast system are generally active, so they are purified in a mild way. Secretory expression proteins are conducive to purification, which can be precipitated with ammonium sulfate, and then further purified by ion exchange, gel filtration chromatography, hydrophobic chromatography and other methods.
The specific methods and operations should be selected according to the properties of the target protein being processed.
