Structural comparison of the <i>Pichia pastoris</i> alcohol oxidase genes

Koutz, P J; Davis, G. R.; Stillman, C A; Barringer, Kevin J.; Cregg, James M.; Thill, G P

doi:10.1002/yea.320050306

Cited by 132 publications

(76 citation statements)

References 26 publications

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“…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

1,097

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: The Pichia Expression Systemsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

View full text Add to dashboard Cite

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“…Expression of the AOX1 gene is tightly regulated and induced by methanol to high levels, typically ≥ 30 % of the total soluble protein in cells grown with methanol as the carbon source. The AOX1 gene has been isolated and the AOX1 promoter is used to drive expression of the gene of interest (Ellis et al, 1985, Koutz et al, 1989.…”

Section: Methylotrophic Yeast Pichia Pastorismentioning

confidence: 99%

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Zhang¹,

Li²,

Agblevor³

2005

Process Biochemistry

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The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (k L a) and subsequently increased the cell mass and protein production compared to conventional fermentation.Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in the conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. The volumetric oxygen transfer coefficient k L a was 1011.9 h -1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h -1 ) but was similar to the conventional 750 rpm (1098 h -1 ). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging.Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. ACKNOWLEDGMENTS

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“…To avoid the toxic effect of hydrogen peroxide, this metabolism occurs at a specialized cell organelle, called peroxisome. In this organelle, the toxic by-products sequestered away from the cell [17,20].…”

Section: Resultsmentioning

confidence: 99%

“…While, low levels of methanol may not be enough to initiate transcription. However, optimal concentration of methanol was necessary to have the balance between slightest toxic effect and highest protein expression [17,[20][21][22][23].…”

Section: Resultsmentioning

confidence: 99%

EFFECT OF METHANOL INDUCTION AND INCUBATION TIME ON EXPRESSION OF HUMAN ERYTHROPOIETIN IN METHYLOTROPIC YEAST Pichia pastoris

Santoso¹,

Herawati²,

Rubiana³

2012

MST

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Erythropoietin (EPO) is a glycoprotein hormone consists of 165 amino acids and has molecular mass of 30,400 Daltons. The large quantities of these hormone required to satisfy clinical demand are currently met by recombinant expression in mammalian cell, namely chinese hamster ovary (CHO). Pichia pastoris has become popular yeast based protein production systems to substitute mammalian expression systems. P. pastoris is capable to use methanol as sole carbon and energy source. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the hEPO gene in methylotropic yeast P. pastoris, strain X33. The present work was carried out to study the optimal methanol concentration for induction and the incubation time to obtain rhEPO protein. To perform this study, the transformed P. pastoris was induced with various concentrations of methanol (0%, 0.5%, 1%, 2.5%, 5%, 10%, and 20%) and incubation times (0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours). The results demonstrate that the highest protein expression level occurred at concentration of 2.5% methanol induction, while the optimal incubation time was at 48 hrs. Abstrak Pengaruh Induksi Methanol dan Waktu Inkubasi terhadap Ekspresi Eritropoietin Manusia pada KhamirMetilotropik Pichia pastoris. Eritropoietin (EPO) adalah hormon glikoprotein yang terdiri dari 165 asam amino dan memiliki berat molekul sebesar 30.400 Daltons. Sebagian besar kebutuhan EPO didapatkan dari hasil sintesis pada sel mamalia Chinese hamster ovary (CHO). Pichia pastoris adalah sejenis khamir yang populer digunakan untuk menggantikan sistem ekspresi pada sel mamalia. P. pastoris dapat menggunakan metanol sebagai satu-satunya sumber energi karbon. Pada studi ini, protein rekombinan EPO (rhEPO) disintesis dengan cara mengekspresikan gen hEPO pada khamir metilotropik P. pastoris strain X33. Studi ini dilakukan untuk mengetahui konsentrasi metanol dan waktu inkubasi yang optimal untuk mensintesis rhEPO. Pada studi ini konsentrasi metanol yang digunakan adalah 0%, 0.5%, 1%, 2.5%, 5%, 10%, dan 20%. Sedangkan waktu inkubasi yang digunakan adalah 0 jam, 24 jam, 48 jam, 72 jam, 96 jam, 120 jam, and 144 jam. Hasil penelitian ini menunjukkan bahwa ekspresi protein yang tertinggi terjadi pada konsentrasi metanol sebesar 2.5% dan waktu inkubasi selama 48 jam.

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Structural comparison of the Pichia pastoris alcohol oxidase genes

Cited by 132 publications

References 26 publications

Heterologous protein production using the Pichia pastoris expression system

Heterologous protein production using the Pichia pastoris expression system

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

EFFECT OF METHANOL INDUCTION AND INCUBATION TIME ON EXPRESSION OF HUMAN ERYTHROPOIETIN IN METHYLOTROPIC YEAST Pichia pastoris

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