Recent Advances in the Expression of Foreign Genes in Pichia pastoris

Cregg, James M.; Vedvick, Thomas S.; Raschke, William C.

doi:10.1038/nbt0893-905

Cited by 771 publications

(502 citation statements)

References 27 publications

Supporting

Mentioning

473

Contrasting

Unclassified

Order By: Relevance

“…More information about the expression vectors available for this system can be found in Cereghino and Cregg [18], Cregg et al [28], Koutz et al [61] and Sreekrishna et al [103]. More recent developments in the use of Pichia expression vectors are discussed below.…”

Section: The Pichia Expression Systemsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

View full text Add to dashboard Cite

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

show abstract

Section: The Pichia Expression Systemsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

-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

show abstract

“…The methylotrophic yeast, Pichia pastoris has been described as a particularly efficient host for high level expression of heterologous genes when these are placed under control of the host alcohol oxidase-1 (AOX1) promoter [4]. The activity of the AOX1 promoter is tightly regulated.…”

Section: Introductionmentioning

confidence: 99%