High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris

Cregg, J. M.; Tschopp, Juerg; Stillman, C A; Siegel, Robert S.; Akong, M.; Craig, William S.; Buckholz, Richard G.; Madden, Knut R.; Kellaris, P. A.; Davis, G. R.; Smiley, Bob L.; Cruze, John A.; Torregrossa, Roberta; ecedil, G. Velic; lebi,; Thill, G P

doi:10.1038/nbt0587-479

Cited by 261 publications

(125 citation statements)

References 39 publications

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“…After 4 days of methanol induction, the yeast cells were pelleted and disrupted with microfluidizer (model 110S ; Microfluidics Corp.), and the cellular debris was eliminated by centrifugation. The rHBsAg was purified from cell lysate by serial ultracentrifugation in caesium chloride and sucrose gradients (Cregg et al, 1987). The gradient fractions containing HBsAg were identified by a sandwich ELISA assay (Surase B-96 EIA, General Biologicals Corp.) which utilized a monoclonal anti-HBs antibody of the IgM type to capture rHBsAg and a conjugated polyclonal anti-HBs from guinea-pig to detect HBsAg.…”

Section: Methodsmentioning

confidence: 99%

Altered antigenicity of 'a' determinant variants of hepatitis B virus.

Chiou

Lee

Kuo

et al. 1997

Journal of General Virology

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Section: Methodsmentioning

confidence: 99%

Altered antigenicity of 'a' determinant variants of hepatitis B virus.

Chiou

Lee

Kuo

et al. 1997

Journal of General Virology

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“…Since the cells must then rely on the weaker AOX2 for methanol metabolism, a slower growing and slower methanol utilization strain is produced. Mut s strains have been found to be advantageous for production of hepatitis B surface antigen [26]. The Mut − , or methanol utilization minus phenotype, are unable to grow on methanol, since these strains have both AOX genes deleted.…”

Section: Methanol Utilization Phenotypesmentioning

confidence: 99%

“…The Mut − , or methanol utilization minus phenotype, are unable to grow on methanol, since these strains have both AOX genes deleted. One of the advantages of this phenotype is that low growth rates may be desirable for production of certain recombinant products [26,27]. Currently, the majority of researchers use the Mut + phenotype [47,102,129], although some researchers are also using the Mut s phenotype [1,71,83,85,126].…”

Section: Methanol Utilization Phenotypesmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

633

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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.

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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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High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris

Cited by 261 publications

References 39 publications

Altered antigenicity of 'a' determinant variants of hepatitis B virus.

Altered antigenicity of 'a' determinant variants of hepatitis B virus.

Heterologous protein production using the Pichia pastoris expression system

Development of vaccines for prevention of botulism

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