Foreign gene expression in yeast: a review

Romanos, Michael A.; Scorer, Carol A.; Clare, Jeffrey J.

doi:10.1002/yea.320080602

Cited by 938 publications

(613 citation statements)

References 389 publications

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“…The yeast strain Pichia pastoris, originally developed by the Phillips Petroleum Company, was chosen as a host because of the high level of recombinant expression exhibited by this system with other proteins [55][56][57][58]. Clare and coworkers [59] reported yields of 12 g/L of TeNT(H C ) when expressed in P. pastoris.…”

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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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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“…In the context of biosensors, complete knowledge of active promoter regions must also be ascertained to effectively express a reporter using this system. Finally, plasmid loss remains a concern, as hosts must be maintained under constant selective pressure (Stearns et al, 1990;Rose and Broach, 1991;Romanos et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Construction of a cytosolic firefly luciferase reporter cassette for use in PCR‐mediated gene deletion and fusion in Saccharomyces cerevisiae

Ainsworth

Rome

Hjortsø

et al. 2012

Yeast

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Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(ÀSKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.

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“…Examples are methanol-utilizing yeasts such as Hansenula polymorpha and Pichiapastoris and the lactose-utilizing species Kluyveromyces lactis and K. marxianus (Romanos et al 1992). Several arguments have been put forward to use 'non-Saccharomyces yeasts' as hosts for heterologous gene expression, including broader substrate specificity, availability of strong inducible promoters, absence of aerobic alcoholic fermentation (i.e.…”

Section: Introductionmentioning

confidence: 99%

Kinetics of growth and sugar consumption in yeasts

Dijken¹,

Weusthuis²,

Pronk³

1993

Antonie van Leeuwenhoek

186

112

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An overview is presented of the steady-and transient state kinetics of growth and formation of metabolic byproducts in yeasts. Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called 'Crabtree effect' probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect in S. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast. S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.'Non-Saccharomyces' yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharornyces yeasts are therefore not suitable for largescale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeast Candida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.

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Foreign gene expression in yeast: a review

Cited by 938 publications

References 389 publications

Development of vaccines for prevention of botulism

Development of vaccines for prevention of botulism

Construction of a cytosolic firefly luciferase reporter cassette for use in PCR‐mediated gene deletion and fusion in Saccharomyces cerevisiae

Kinetics of growth and sugar consumption in yeasts

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