Pichia pastoris

Taxobox | name = "Pichia pastoris"
regnum = Fungi
phylum = Ascomycota
classis = Saccharomycetes
ordo = Saccharomycetales
familia = Saccharomycetaceae
genus = "Pichia"
species = "P. pastoris"
binomial = "Pichia pastoris"
binomial_authority = (Guillierm.) Phaff 1956

"Pichia pastoris" is a species of methylotrophic yeast. "Pichia" is widely used for protein expression using recombinant DNA techniques. Hence it is used in biochemical and genetic research in academia and the biotechnical industry.

"Pichia pastoris" as an expression system

"Pichia pastoris" is frequently used as an expression system for the production of proteins. A number of properties makes "Pichia" suited for this task: "Pichia" has a high growth rate and is able to grow on a simple, inexpensive medium. "Pichia" can grow in either shake flasks or a fermenter, which makes it suitable for both small and large scale production.

"Pichia pastoris" has two alcohol oxidase genes, AOX1 and AOX2, which have a strongly inducible promoter. These genes allow "Pichia" to use methanol as a carbon and energy source. The AOX promoters are induced by methanol and are repressed by e.g. glucose. Usually the gene for the desired protein is introduced under the control of the AOX1 promoter, which means that protein production can be induced by the addition of methanol. In a popular expression vector, the desired protein is produced as a fusion product to the secretion signal of the α-mating factor from "Saccharomyces cerevisiae" (baker's yeast). This causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. There are commercially available plasmids, that have these features incorporated.

Comparison to other expression systems

In standard molecular biology research, the bacterium "Escherichia coli" is the most frequently used organism for production of recombinant DNA and proteins. This is due to "E. coli's" fast growth rate, good protein production rate and undemanding growth conditions. Protein expression in "E. coli" is usually faster than in "Pichia pastoris" for several reasons: Competent "E. coli" cells can be stored frozen, and thawed immediately before use, whereas "Pichia" cells have to be produced immediately before use. Expression yields in "Pichia" vary between different clones Fact|date=June 2008, and usually a large number of clones needs to be screened for protein expression before a good producer is found. Optimal induction times of "Pichia" are usually on the order of days, whereas "E. coli" usually reaches optimal yields within hours of induction. The major advantage of "Pichia" over "E. coli" is that "Pichia" is capable of producing disulfide bonds and glycosylations in proteins. This means that in cases where disulfides are necessary, "E. coli" might produce a misfolded protein, that is usually inactive or insoluble.

The well-studied "Saccharomyces cerevisiae" (baker's yeast) is also used as an expression system with similar advantages over "E. coli" as "Pichia". However "Pichia" has two main advantages over "S. cerevisiae" in laboratory and industrial settings:

Firstly, "Pichia", as mentioned above, is a methylotroph, meaning it can grow with the simple alcohol methanol as its only source of energy — "Pichia" can easily be grown in cell suspension in reasonably strong methanol solutions that would kill most other micro-organisms, a system that is cheap to set up and maintain.

Secondly, "Pichia" can grow to very high cell densities, and under ideal conditions can multiply to the point where the cell suspension is 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.

Compared to other expression systems such as S2-cells from "Drosophila melanogaster" or Chinese Hamster Ovary cells, "Pichia" usually gives much better yields. Cell lines from multicellular organisms usually require complex rich media, including amino acids, vitamins and growth factors. These media significantly increase the cost of producing recombinant proteins. Additionally, since "Pichia" can grow in media containing only one carbon source and one nitrogen source, it is suitable for isotopic labelling applications in e.g. protein NMR. However, a number of proteins require chaperonins for proper folding. Thus, "Pichia" is unable to produce a number of proteins for which the host lacks the appropriate chaperones. Recently a group has managed to create a strain that produces EPO in its normal glycosylation form ( [http://www.sciencemag.org/cgi/content/abstract/313/5792/1441 Hamilton SR. et al, Science, Sept 8, 2006] ). This was achieved by exchanging the enzymes responsible for the fungal type glycosylation, with the mammalian homologs. Thus, the altered glycosylation pattern allowed the protein to be fully functional.

"Pichia pastoris" as a model organism

Another advantage of Pichia pastoris is its similarity to the well-studied "Saccharomyces cerevisiae" (also known as baker's yeast). As a model organism for biology, and having been used by man for various purposes throughout history, "S. cerevisiae" is well studied, to say the least. The two yeast species ("Pichia", "Saccharomyces") have similar growth conditions and tolerances, and thus the culturing of "Pichia pastoris" can be readily adopted by labs without specialist equipment.