Yeast Expression System and Method

The Production of Yeast Expression System

The development of genetic engineering technology has shown broad prospects for the synthesis and production of foreign proteins by microorganisms. For a long time, people have used E. coli as a host to express a variety of proteins. This is because E. coli has several advantages, such as clear genetic background and biochemical characteristics, easy operation, rapid growth and simple nutritional requirements.
However, the E. coli expression system has several defects: A: lack of post-translational modification and processing of eukaryotic proteins, such as shearing, glycosylation, formation of disulfide bonds, etc.; B: most expressed proteins exist in the form of inclusion bodies, which require Only after complex renaturation can the conformation and activity be restored; C: There are many background contaminants and it is troublesome to purify; D: The expression level is generally not very high.

Since 1979, in order to overcome the shortcomings of the E. coli expression system, the yeast expression system has been developed. Saccharomyces cerevisiae was the first to be used, because Saccharomyces cerevisiae has been used in the brewing industry and the bakery industry for thousands of years, and is considered a safe organism; in addition, yeast is a single-celled lower eukaryotic organism, which has the easy cultivation and reproduction of prokaryotes. It is fast and convenient for genetic engineering operations. It also has the function of eukaryotic protein post-translational processing. It has an intracellular environment and sugar chain processing system suitable for the correct folding of eukaryotic gene products. It can also secrete foreign proteins to In the culture medium, it is conducive to purification and can reduce the metabolic load of the host cell. Especially due to the discovery of the 2u plasmid of Saccharomyces cerevisiae and the breakthrough of yeast transformation technology, the genetic engineering expression system of Saccharomyces cerevisiae was established and applied.

In 1981, Hizeman et al. reported for the first time that the recombinant human interferon gene was expressed in Saccharomyces cerevisiae and succeeded.

Composition of Yeast Expression System

Host: Saccharomyces cerevisiae, fission yeast, Kluyvera lactis, Pichia pastoris, etc.

Plasmid Vector

• Plasmid Type:

  • Yeast replicating plasmid, YRp

  • Yeast centromeric plasmid, YCp

  • Yeast episomal plasmid, YEp

  • (Autonomous replication, high copy number, unstable, easy to lose)

  • Yeast integrating plasmid (YIp) (Good stability, low copy number)

  • Yeast artificial chromosome

• Selection Markers:

  • A. auxotrophic selection markers (leucine synthase gene leu2, tryptophan synthase gene tril, urine synthase gene ura3 histamine synthase gene his3)

  • B dominant selection marker (amino acid glycoside antibiotic G418, chlorenzyme, hygroenzyme)

• Related elements of foreign gene expression: promoter (pgk1, AOX, LAC4), terminator secretion signal sequence

Main Yeast Expression System

• Saccharomyces cerevisiae expression system

  • Saccharomyces cerevisiae

• Kluvia lactis expression system

  • Kluyveromyces lactis

• Methanol-trophic yeast expression system

  • Candida bodinii

  • Hansenula polymorph

  • Pschia methanolica

  • Pichia pastoris

• Schizo saccharomyces pombe

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