Pichia pastoris
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Pichia pastoris is a species of yeast that is useful for recombinant genetic research. It is a methylotroph.
In standard cell culture and in transgenics, the bacterium Escherichia coli is used by far the most, due to fast growth rate, good protein production rate and undemanding growth conditions. However, there are several disadvantages to using E. coli for the production of transgenic proteins.
Among these is the fact that, as a prokaryote, E. coli cannot glycosylate proteins, a step often taken in eukaryotes post-translationally. Many times, this results in a mis-folded, non-functional protein that may be swiftly eradicated by a host upon introduction. This makes it unusable in vivo, or at least inefficient.
Pichia pastoris is well suited to protein expression due to a high growth rate and a convenient expression system already present in the microbe (See the information on "alcohol oxidases", below). Additionally, as a eukaryote, P. pastoris post-translational modifications are more similar to human protein modifications, and P. pastoris is able to produce disulphide bonds.
Another advantage of Pichia pastoris is its similarity to the well-studied Saccharomyces cerevisiae, also known as bakers yeast. As a model organism for biology, and having been used by humanity for various purposes throughout history, S. cerevisiae is well studied, to say the least. The two yeast species (Pichia, Saccharomyces) have similar growth conditions (With one important difference, below) and tolerances, and thus culturing of Pichia pastoris can be readily adopted by labs without specialist equipment.
However there are other, specific reasons why P. pastoris is of use. With the above reasoning, why not simply use Saccharomyces cerevisiae? Indeed, S. cerevisiae sees extensive use, however Pichia has two main advantages that set it apart.
Firstly, Pichia, as mentioned above, is a methylotroph, meaning it can grow and thrive on the simple alcohol methanol alone, whereas other microbes would die from alcohol poisoning. Pichia can be grown in cell suspension in reasonably strong methanol solution without difficulty, which is very cheap to set up and maintain.
Secondly, Pichia can grow to startlingly high cell densities, and under ideal conditions can multiply to practically a paste. As the protein yield from expression in a microbe is roughly equal to the product of the protein produced per cell and the number of cells, this makes Pichia of great use when trying to produce large quantities of protein without expensive equipment.
Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2, which have a strong inducible promoter. When a gene is placed after this promotor, it is not produced in aerobic conditions, and P. pastoris can grow without being influenced by toxicity of the recombinant gene product. When put in an anaerobic environment, the protein will be produced. There are plasmids available which facilitate the placement of the genes after these promoters.
Recently a group has managed to create a strain that produces EPO in its normal glycosilation form. This was achieved by exchanging the enzymes responsible for the fungal type glycosilation, with the mammalian homologs. Thus the glycosilation patern allowed the protein to be fully functional.
