Yeast Expression Technologies
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Yeast Expression System
Yeast is a eukaryotic organism and has some advantages and disadvantages over E. coli. One of the major advantages is that yeast cultures can be grown to very high densities, which makes them especially useful for the production of isotope labeled protein for NMR. The two most used yeast strains are Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris.
Various yeast species have proven to be extremely useful for expression and analysis of eukaryotic proteins. These yeast strains have been genetically well characterized and are known to perform many posttranslational modifications. These single-celled eukaryotic organ-isms grow quickly in defined medium, are easier and less expensive to work with than insect or mammalian cells, and are easily adapted to fermentation. Yeast expression systems are ideally suited for large-scale production of recombinant eukaryotic proteins.
In some instances the most cost-effective expression of functional enzymes is the yeast expression system. The major advantages of yeast expression system are:
- High yield
- High productivity
- Chemically defined media
- Product processing similar to mammalian cells
- Stable production strains
- Lower protein production cost
More specifically, yeast expression system has the following merits or strengths:
Yeast is an established industrial fermentation system and supports high-level recombinant protein production. The high-level protein production can be reached by taking care of the following factors:
- Adequate copies of vector (10-100 copies per cell)
- Suitable promoters
- Proper inducible system
- Targeted cellular location
High Cell Densities
When yeast is grown with the high-cell-density fermentation technology, unprecedented levels of cell mass per liter of fermentation fluid are produced. The system has attained dry-cell-weight densities exceeding 100 gram/liter and continuous fermentation productivities of 10 to 12 grams of recombinant protein/liter/hour.
The growth medium that feeds yeast is completely defined. It consists of a simple, inexpensive formulation. The carbon source is fed to the fermentor at a rate designed to achieve maximum cell density while maintaining optimal production of foreign protein. This process minimizes any toxic effects the foreign protein might have on the yeast.
As a eukaryotic system, the Yeast Expression System produces mammalian-like proteins. For example, the expression of Hepatitis B surface antigen (HBsAg) in yeast leads to production of particles that are immunoreactive with anti-HBsAg antibodies. These particles are similar to Dane particles isolated from the sera of human carriers.
Generations of Stability
Expression of foreign genes is achieved by integration of foreign DNA into the chromosomal DNA of host genome. The integrated DNA is stable for many generations; all cells can produce the protein. In contrast, plasmid-based systems require selective pressure on plasmids to maintain the foreign DNA. Cells that lose the plasmid cannot produce the desired foreign protein.
The Yeast Expression System requires no special handling. It was developed to withstand the adverse conditions of large scale, continuous fermentors. This feature makes yeast able to survive unexpected disruptions in the fermentation process.
High per-cell expression levels combined with high cell-density growth of yeast translates into greater quantities of recombinant protein per fermentor volume. This reduces production costs by increasing the amount of product per fermentation run.
Protein purification is another cost-saving area. The yeast system can secrete protein into the medium, so the broth that enters purification contains a higher concentration of the desired protein. Pure protein is recovered with higher yield and lower cost.
GenWay has successfully applied the yeast expression system for producing various proteins. One of the examples is expressing recombinant sweet protein (Sweetin™). The natural product of Sweetin™, known as monellin, is a heterodimer and can be isolated from "Serendipity Berries" of the West African Plant. To obtain stable and secretable large-scale production, two chains of the monellin molecule were linked together and expressed in yeast as a single chain recombinant protein, Sweetin™. The core technology for producing Sweetin™ is a high-density yeast expression system (HIDYES). The fermentation process allows secretion of the product into culture broth, which make the protein purification process highly cost and time-effective.
Recombinant protein SweetinTM production in yeast.
A. Cell density and SweetinTM production curves recorded in yeast fermentation. B. SDS-PAGE analysis of fractions of SweetinTM protein purification. Lanes 1-5: Molecular
Weight Markers in kilo-Dalton (kDa); Crude product in the fermentation broth (5 ul loading); partial purification of the product with simple sizing filtration; purified product SweetinTM through a column processing; purified natural product (monellin) from Sigma Co. (1 ug loading).
For other systems of protein expression, please visit the pages:
GenWay offers comprehensive custom services in recombinant protein expression.
For more detailed information, please see the specific description pages in protein expression