Expression of Recombinant Proteins in Yeast - - BioPharm International

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Expression of Recombinant Proteins in Yeast


Table 1. Saccharomyces Plasmids.
Yeast selection markers can be classified into two types: complementation markers and dominant selection markers. Dominant selection markers are antibiotic markers that can be used in yeast such as G418 and cyclohexamide. Complementation markers are marker genes that complement an auxotrophic mutation in the genome like URA3, TRPI, HIS3, and LEU2. These auxotrophic markers are used in selection of recombinants with all three types of expression systems (integration, episomal, and centromeric plasmids). A summary of the various plasmids and their selection systems is shown in Table 1.

Promoters


Table 2. Saccharomyces Promoters.
A range of yeast promoters is available for protein expression. Some like ADH2,SUC2 are inducible and others like GAPDH are constitutive in expression. Similar to the selection markers, a wide variety of combinations of promoters, markers, and expression systems are commercially available (Table 2).

Secretion Signals

A variety of secretion signals are also available for expression in S. cerevisiae. These include:

  • Prepro alpha factor12
  • HSp15012
  • PHO112
  • SUC212
  • KILM1 (killer toxin type 1)12
  • GGP113

These systems and combinations of promoter, vectors, and signal sequence can be used for high-level expression of recombinant proteins in yeast.

Pichia Pastoris

Pichia pastoris, a methyltrophic yeast that grows to high cell density in a defined medium, is capable of a high level of recombinant gene expression from a tightly controlled promoter, and capable of several post-translational modifications performed by higher eukaryotes.14 Alcohol oxidase (AOX) catalyzes the first step in metabolism of methanol, and because alcohol oxidase has a low affinity for O2, the yeast expresses large amounts of alcohol oxidase, up to 30% of total cell protein, to compensate.14,15 Thus, it was determined that utilization of this promoter in the presence of methanol could lead to high recombinant protein production. The glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter has since been developed for constitutive expression of non-toxic proteins in Pichia.16 The promoter for the Pichia glutathione-dependent formaldehyde dehydrogenase (fld) gene is also available as an inducible promoter with induction by methyl-amine.17 Higgins and Cregg list numerous proteins that have been produced in Pichia.14


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