Pichia pastoris 2202156 220373893 2008-06-19T15:29:21Z SiameseTurtle 4597939 /* Pichia pastoris as an expression system */ {{Unreferenced|date=December 2006}} {{Taxobox | name = ''Pichia pastoris'' | regnum = [[Fungi]] | phylum = [[Ascomycota]] | subphylum = [[Saccharomycotina]] | classis = [[Saccharomycetes]] | ordo = [[Saccharomycetales]] | familia = [[Saccharomycetaceae]] | genus = ''[[Pichia]]'' | species = '''''P. pastoris''''' | binomial = ''Pichia pastoris'' }} '''''Pichia pastoris''''' is a species of [[methylotroph]]ic [[yeast]]. ''Pichia'' is widely used for [[protein expression]] using [[recombinant DNA]] techniques. Hence it is used in [[biochemistry|biochemical]] and [[Genetics|genetic]] research in [[academia]] and the [[biotechnology|biotechnical industry]]. ==''Pichia pastoris'' as an expression system== ''Pichia pastoris'' is frequently used as an [[gene expression#expression system|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 [[regulation of gene expression|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 [[plasmid]]s, 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 cell|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 [[Cloning|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 [[glycosylation]]s 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 cell]]s, ''Pichia'' usually gives much better yields. Cell lines from multicellular organisms usually require complex rich media, including [[amino acids]], [[vitamin]]s and [[growth factor]]s. 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 [[chaperonin]]s for proper folding. Thus, ''Pichia'' is unable to produce a number of proteins for which the host lacks the appropriate chaperones. Additionally ''Pichia'' has been reported to produce hyperglycosylations of a number of proteins (citation needed), which makes it unsuitable for some applications such as [[structural biology]]. Recently a group has managed to create a strain that produces [[Erythropoietin|EPO]] in its normal glycosylation form {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. [[Category:Ascomycota]] [[Category:Model organisms]] [[Category:Yeasts]]