Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris

Sreekrishna, K.; Brankamp, Robert G.; Kropp, K; Blankenship, Dale T.; Tsay, Jiu-Tsair; Smith, Phil L.; Wierschke, Jonathan D.; Subramaniam, Arun; Birkenberger, Lori A.

doi:10.1016/s0378-1119(96)00672-5

Cited by 398 publications

(258 citation statements)

References 27 publications

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“…These strains have a disruption in the genes encoding proteinase A (PEP4 ) and/or proteinase B (PRB1 ) [104]. Proteinase A is a vacuolar aspartyl protease necessary for the activation of vacuolar proteases, such as carboxypeptidase Y and proteinase B. Proteinase B has about half the activity of the processed enzyme before being activated by proteinase A.…”

Section: Cell-level Strategiesmentioning

confidence: 99%

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Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

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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Section: Cell-level Strategiesmentioning

confidence: 99%

“…The use of yeast extract and casamino acids increased protein secretion and accumulation [104], and L-arginine and EDTA increased accumulation of a single-chain antibody (scFv) three-to five-fold, although no expression levels were mentioned specifically [98].…”

Section: Nutrient Considerationsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

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“…We prepared synthetic genes for the H C of BoNT with codon usage specified by highly expressed genes in E. coli and yeast. Genes designed and constructed using E. coli codon usage [62] expressed full-length products in both E. coli and P. pastoris, while genes designed and constructed using P. pastoris codon usage [63] likewise, expressed proteins with the expected molecular mass in both hosts.…”

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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“…4). Several previous reports have proposed that the longer the 5′UTR, the greater the chance that translation initiation complex will disassociate from the mRNA as it scans from the cap to the first AUG (Sreekrishna, 1993; Tuller et al, 2009). This would explain an incremental drop off in β-galactosidase with additions of 20–50 nucleotides.…”

Section: Resultsmentioning

confidence: 99%

“…Both of these actions lengthen the 5′UTR. Although it was reported that an increase of 36 nucleotides in the AOX1 5′UTR reduced the expression of human serum albumin by about 50-fold (Sreekrishna, 1993), no systematic analysis has been performed to correlate the effect of incremental 5′UTR extension with protein expression levels.…”

Section: Introductionmentioning

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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Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris

Cited by 398 publications

References 27 publications

Heterologous protein production using the Pichia pastoris expression system

Heterologous protein production using the Pichia pastoris expression system

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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