Pichia pastoris as a host system for transformations.

Cregg, J. M.; Barringer, Kevin J.; Hessler, Anita Y.; Madden, Knut R.

doi:10.1128/mcb.5.12.3376

Cited by 469 publications

(320 citation statements)

References 25 publications

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“…The P. pastoris GS115 strain (Invitrogen) is derived from the wild-type strain NRRL-Y 11430 (Northern Regional Research Laboratories). It has a mutation in the histinol dehydrogenase gene (HIS4) and was generated by nitrosoguanidine mutagenesis at Phillips Petroleum Co 35 . It is the most frequently used Pichia strain for heterologous protein production.…”

Section: Methodsmentioning

confidence: 99%

Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

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The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production. Despite its biotechnological importance, relatively few genetic tools or engineered strains have been generated for P. pastoris. To facilitate progress in these areas, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris. We also provide manually curated annotation for its 5,313 protein-coding genes.The methylotrophic yeast Pichia pastoris is by far the most commonly used yeast species in the production of recombinant proteins 1 and is employed in laboratories around the world to produce proteins for basic research and medical applications. It is also an important model organism for the investigation of peroxisomal proliferation and methanol assimilation. The P. pastoris expression technology has been commercially available for many years. P. pastoris grows to high cell density, provides tightly controlled methanol-inducible transgene expression and efficiently secretes heterologous proteins in defined media. Several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin 2 ) or for which glycosylation is needed only for proper folding (such as several vaccines 3 ) are already on the market. An important recent breakthrough has been the development of P. pastoris strains with humantype N-glycosylation [4][5][6] . Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 . Moreover, monoclonal antibodies can be made at gramper-liter scale in the humanized glycosylation-homogenous strains 8 .For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.

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Section: Methodsmentioning

confidence: 99%

Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

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“…The expression cassette was integrated into the chromosomal alcohol oxidase of P. pastoris strain GS115 [59]. Yeast transformants expressing the selectable markers, histidine dehydrogenase [70] and aminoglycoside phosphotransferase 3' I [68], were isolated. Clones were assessed by their ability to express recombinant proteins with expected molecular masses as judged by SDS-PAGE and Western blot analysis.…”

Section: Design Of Synthetic Bont(h C ) Gene Construct and Expressimentioning

confidence: 99%

Development of vaccines for prevention of botulism

2000

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-Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum. This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis. The development of vaccines that protect against botulism dates back to the 1940s. Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines. Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT. The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C. botulinum neurotoxins (rBoNT(H C )). The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells. Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates. A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS botulinum neurotoxin / vaccine / C-fragment / purification / efficacy

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“…yJC100 cells were cultured in either YPD medium (1% yeast extract, 2% peptone, and 2% glucose), YND (minimal medium with 1% dextrose) or YNM (minimal medium with 0.5% methanol) (Cregg and Madden, 1988; Cregg et al, 1985). …”

Section: Methodsmentioning

confidence: 99%

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

Staley

Huang

Nattestad

et al. 2012

Gene

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Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging.

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Pichia pastoris as a host system for transformations.

Cited by 469 publications

References 25 publications

Genome sequence of the recombinant protein production host Pichia pastoris

Genome sequence of the recombinant protein production host Pichia pastoris

Development of vaccines for prevention of botulism

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

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