PrepTutorEJDIC

(教会・教会員を世話する)牧師

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2013/10/06 09:58:55」(JST)

wiki en

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[edit]

Pichia pastoris is frequently used as an expression system for the production of proteins. A number of properties makes P. pastoris suited for this task: P. pastoris has a high growth rate and is able to grow on a simple, inexpensive medium. P. pastoris 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.^[1] 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 (such as the pPICZα vector).^[2]

Comparison to other expression systems[edit]

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, 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.^[3] This means that in cases where disulfides are necessary, E. coli might produce a misfolded protein, that is usually inactive or insoluble.^[4]

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.^[3]

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.^[3] 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. In 2006 a research group has managed to create a strain that produces Erythropoietin in its normal glycosylation form.^[5] 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[edit]

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.

The Pichia pastoris GS115 genome has been sequenced by the Flanders Institute for Biotechnology (VIB) and Ghent University (UGent) and published in Nature Biotechnology.^[6] The genome sequence and gene annotation can be browsed through the BOGAS system.

References[edit]

^ Daly R, Hearn MT (2005). "Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production". J. Mol. Recognit. 18 (2): 119–38. doi:10.1002/jmr.687. PMID 15565717.
^ "Description of pPICZα vector by its vendor Invitrogen".
^ ^a ^b ^c Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009). "Expression in the yeast Pichia pastoris". Meth. Enzymol. Methods in Enzymology 463: 169–89. doi:10.1016/S0076-6879(09)63013-5. ISBN 978-0-12-374536-1. PMID 19892173.
^ Brondyk WH (2009). "Selecting an appropriate method for expressing a recombinant protein". Meth. Enzymol. Methods in Enzymology 463: 131–47. doi:10.1016/S0076-6879(09)63011-1. ISBN 978-0-12-374536-1. PMID 19892171.
^ Hamilton SR, Davidson RC, Sethuraman N, et al. (September 2006). "Humanization of yeast to produce complex terminally sialylated glycoproteins". Science 313 (5792): 1441–3. doi:10.1126/science.1130256. PMID 16960007.
^ De Schutter K., Lin Y.-C., Tiels P, et al. (2009). "Genome sequence of the recombinant protein production host Pichia pastoris". Nature Biotechnology 27 (6): 561–566. doi:10.1038/nbt.1544. PMID 19465926.

UpToDate Contents

全文を閲覧するには購読必要です。 To read the full text you will need to subscribe.

1. 新生児における特殊な真菌感染症 unusual fungal infections in the neonate

English Journal

Recombinant snake venom metalloproteinase inhibitor BJ46A inhibits invasion and metastasis of B16F10 and MHCC97H cells through reductions of matrix metalloproteinases 2 and 9 activities.

Ji MK, Shi Y, Xu JW, Lin X, Lin JY.SourceaKey Laboratory of Ministry of Education for Gastrointestinal Cancer, Research Center of Molecular Medicine bDepartment of Dermatology, First Affiliated Hospital, Fujian Medical University cDepartment of Molecular Pathology, Fujian Provincial Tumor Hospital, The Teaching Hospital of Fujian Medical University, Fuzhou, China.
Anti-cancer drugs.Anticancer Drugs.2013 Jun;24(5):461-72. doi: 10.1097/CAD.0b013e32835f258d.
Studies have shown that the recombinant BJ46a (rBJ46a) protein can reduce matrix metalloproteinase (MMP) activities and inhibit invasion and metastasis of melanoma cells. Here, we optimized the Pichia pastoris system to evaluate rBJ46a protein as an anticancer agent. The Enzchek gelatinase/collagena
PMID 23442578

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris.

Lin-Cereghino GP, Stark CM, Kim D, Chang J, Shaheen N, Poerwanto H, Agari K, Moua P, Low LK, Tran N, Huang AD, Nattestad M, Oshiro KT, Chang JW, Chavan A, Tsai JW, Lin-Cereghino J.SourceDepartment of Biological Sciences, University of the Pacific Stockton, CA 95211, USA.
Gene.Gene.2013 May 1;519(2):311-7. doi: 10.1016/j.gene.2013.01.062. Epub 2013 Feb 21.
The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to
PMID 23454485

Japanese Journal

Isolation and Characterization of CcAbf62A, a GH62 α-L-Arabinofuranosidase, from the Basidiomycete Coprinopsis cinerea

Hashimoto Kohsuke,Yoshida Makoto,Hasumi Keiji
Bioscience, Biotechnology, and Biochemistry 75(2), 342-345, 2011
… The recombinant CcAbf62A, expressed in Pichia pastoris, released L-arabinose from both wheat arabinoxylan and oat-spelt xylan. …
NAID 130000406585

Expression and Characterization of Full-Length Ampullaria crossean Endoglucanase EG65s and Their Two Functional Modules

Yin Qiuyu,Teng Yigang,Li Yanhong [他],DING Ming,ZHAO Fukun
Bioscience, Biotechnology, and Biochemistry 75(2), 240-246, 2011
… To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). …
NAID 130000406569

Related Pictures

★リンクテーブル★

「Pichia」

Pichia pastoris
Scientific classification
Kingdom:	Fungi
Phylum:	Ascomycota
Class:	Saccharomycetes
Order:	Saccharomycetales
Family:	Saccharomycetaceae
Genus:	Komagataella
Species:	P. pastoris
Binomial name
	Pichia pastoris; (Guillierm.) Phaff 1956

　　[★] ピキア、ピキア属、Pichia属

関: hansenula

酵母の一属

[pmid15565717-1] Daly R, Hearn MT (2005). "Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production". J. Mol. Recognit. 18 (2): 119–38. doi:10.1002/jmr.687. PMID 15565717.

[Invitrogen-pICZ-2] "Description of pPICZα vector by its vendor Invitrogen".

[pmid19892173-3] Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009). "Expression in the yeast Pichia pastoris". Meth. Enzymol. Methods in Enzymology 463: 169–89. doi:10.1016/S0076-6879(09)63013-5. ISBN 978-0-12-374536-1. PMID 19892173.

[pmid19892171-4] Brondyk WH (2009). "Selecting an appropriate method for expressing a recombinant protein". Meth. Enzymol. Methods in Enzymology 463: 131–47. doi:10.1016/S0076-6879(09)63011-1. ISBN 978-0-12-374536-1. PMID 19892171.

[pmid16960007-5] Hamilton SR, Davidson RC, Sethuraman N, et al. (September 2006). "Humanization of yeast to produce complex terminally sialylated glycoproteins". Science 313 (5792): 1441–3. doi:10.1126/science.1130256. PMID 16960007.

[De_Schutter-6] De Schutter K., Lin Y.-C., Tiels P, et al. (2009). "Genome sequence of the recombinant protein production host Pichia pastoris". Nature Biotechnology 27 (6): 561–566. doi:10.1038/nbt.1544. PMID 19465926.

匿名

検索

案内

案内

Pichia pastoris