Application of Pichia pastoris expression system in medicine

Posted by beauty33 on November 21st, 2019

The Pichia pastoris (P. pastoris) expression system is currently the most commonly used protein expression system after E. coli. It is widely used in laboratory and production scale protein preparation, characterization and structural analysis. There are thousands of proteins have been successfully expressed and purified in the Pichia pastoris system.

The Pichia pastoris expression system has the characteristics of easy high-density fermentation, effective secretion of the product, proper glycosylation, and convenient culture, and has become a very complete exogenous gene expression system. In the field of pharmaceutical proteins, various proteins such as insulin, hepatitis B surface antigen, human serum albumin, and epidermal growth factor have been expressed using P. pastoris and commercialized. A large number of examples have confirmed that the P pastoris expression system is an efficient, practical and simple exogenous gene expression system that can increase the expression level and maintain the biological activity of the product, which is very suitable for expansion to an industrial scale.

1. P pastoris expression system expresses insulin

Diabetes is a clinical syndrome caused by the interaction of genetic and environmental factors. Its basic pathological features are metabolic disorders caused by absolute or relative insulin secretion. As the most important hypoglycemic agent, insulin is effective against all types of diabetes caused by insulin deficiency and has a huge market demand. Therefore, the development of new ways to produce insulin and the optimization of existing methods for producing insulin have always been an important topic in the field of medical biochemistry.

Pichia pastoris is a protein expression system developed in recent years. Its alcohol oxidase gene promoter is an inducible strong promoter that can be used to regulate the expression of foreign proteins. The system has the advantages of simple operation technique, high level of intracellular or extracellular expression, and post-translational modification and processing of the expression product. Genetic recombination technology is used to construct a Pichia methanolica engineering strain that efficiently expresses insulin precursors, fermentative production of human insulin.

2. Hepatitis B surface antigen

Hepatitis B (HB) is a worldwide infectious disease caused by Hepatitis B virus (HBV). Its envelope protein hepatitis B surface antigen HBsAg can induce the body to produce protective antibodies against hepatitis B surface antibody HBsAb. It is the main component of hepatitis B vaccine. At the same time, HBsAg is also the main reagent for the successful diagnosis of immune by ELISA. The preparation of HBsAg in large quantities is of great significance in the prevention and detection of hepatitis B.

HBsAg is the most important and most commonly used marker for the diagnosis of HBV infection. The mutation of the gene region of the "a" determinant causes its HBsAg not to be recognized by monoclonal or polyclonal antibodies in some commercially available diagnostic kits, thus increasing the HBsAg reagent. The clinical sensitivity of the cassette and the ability to recognize mutants are necessary. At present, there are many kinds of HBsAg mutants, among which G145R is the most common and important, and is also the main research object. The Pichia pastoris expression system was established, and the expression and purification of HBsAg and its mutant G145R were carried out. The preliminary study of these two proteins can lay a foundation for further improving the HBsAg diagnostic kit and its mutation detection.

3. Tumor necrosis factor

Studies have shown that the bioactive part of human tumor necrosis factor-related apoptosis ligand (TRAIL) is secreted and expressed in Pichia pastoris. The proteins with molecular weights of approximately 19kD and 38kD in the fermentation broth can be specifically recognized by TRAIL polyclonal antibodies, and having an activity of inducing apoptosis of tumor cells in vitro. Analysis by thin layer scanning showed that the target protein could account for up to 36% of total soluble protein. The experimental results show that TRAIL expressed in the Pichia pastoris expression system can exist as an oligomer and has biological activity.

4. Epidermal growth factor

The Pichia pastoris genetically engineered strain with high expression of recombinant human epidermal growth factor (rhEGF) was constructed and screened.

5. Human serum albumin

Human serum albumin is a protein drug that is widely used in medicine and is in great demand. At present, the method of plasma extraction production is difficult to meet the requirements of the market, and the production of human serum albumin by genetic engineering method has undoubtedly great commercial value. Due to the many advantages of the Pichia pastoris expression system, it has great advantages in expressing foreign proteins. The genetically engineered strains secreting recombinant human serum albumin (rHSA) were constructed and screened with Pichia pastoris, and the fermentation conditions were studied. Immunoblot detection of the expressed rHSA is immunogenic.

6. Antibodies

The human anti-rabies virus full-molecule antibody was expressed in Pichia pastoris by a stepwise integration method. Studies have found that secreted human whole-molecule antibodies with rabies virus antigen-binding activity can be induced by methanol in Pichia pastoris.

Application prospect of Pichia pastoris expression system

(1). Expression of a biologically active functional protein

As a mature system for the expression of heterologous proteins, Pichia pastoris has the characteristics of post-translational processing of eukaryotes that are the most attractive for widespread use.

(2). Expressing medical products

The Pichia pastoris system can be used to produce medically high-efficiency proteins, for example, recombinant proteins produced by Pichia pastoris include bovine trypsinogen, human 2A-type protein phosphatase (PP2A) subunit, hepatitis B virus pre-S1 protein, and hepatitis E, the virus reads frame 2, human interleukin-2, and tumor necrosis factor. In addition, a key protease MT1-MMP of tumor cells, after deleting the C-terminal transmembrane region, transforms yeast and obtains expression, which is of great significance for the treatment of cancer.

(3). Pichia pastoris can be a powerful tool for studying some important theories and research

The expression product of Pichia pastoris can be used for structural studies. The Pichia pastoris expression system expresses 15N-labeled barley non-specific lipid transfer protein type 2 (ns-LTP2) under strict control of physiological, biochemical and ligation conditions. The obtained ns-LTP2 protein can be used for the study of fine structure.

With the further development of yeast genetics and biology, Pichia pastoris has been increasingly used as an expression system for foreign genes. It will play an increasingly important role in the production of functional proteins, the development of commercial products and scientific research.

Further reading: Recombinant Protein Expression in Pichia Pastoris


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