High‐yield secretion of recombinant gelatins by Pichia pastoris

Werten, Marc W. T.; Bosch, Tanja J. van den; Wind, Richèle D.; Mooibroek, H.; Wolf, Frits A. de

doi:10.1002/(sici)1097-0061(199908)15:11<1087::aid-yea436>3.3.co;2-6

Cited by 82 publications

(189 citation statements)

References 30 publications

(37 reference statements)

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“…Fungal protein-expression systems do not suffer from the same limitations, and protein titers of 14.8 and 35 g͞liter have been reported for secreted heterologous proteins in yeast and the filamentous fungus Trichoderma reesei, respectively (3,4). However, glycoproteins derived from fungal expression systems contain nonhuman N-glycans of the high mannose (Man) type, which are immunogenic in humans and thus of limited therapeutic value (5).…”

mentioning

confidence: 99%

“…Fungi and mammals share initial steps of protein N-glycosylation, which involves the site-specific transfer of (Glc) 3 -(Man) 9 -(GlcNAc) 2 from the luminal side of the endoplasmic reticulum (ER) to the de novo synthesized protein by an oligosaccharyltransferase complex. Subsequent trimming by glucosidases I and II and a specific ER-residing ␣-1,2-mannosidase leads to the formation of a (Man) 8 -(GlcNAc) 2 structures (isomer Man8B) (Fig.…”

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confidence: 99%

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Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

Choi

Bobrowicz²,

Davidson³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

325

211

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The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating α-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active α-1,2-mannosidase and human β-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of α-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)5-(GlcNAc)2] activity, we were able to isolate several yeast strains that produce almost homogenous N-glycans of the (Man)5-(GlcNAc)2 type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)5-(GlcNAc)2 in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)5-(GlcNAc)2 as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation

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confidence: 99%

mentioning

confidence: 99%

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

Choi

Bobrowicz²,

Davidson³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

325

211

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“…peptone, casamino acids) to the culture medium, possibly by acting as alternative and competing substrates for one or more problem proteases, and these supplements can also repress protease induction caused by nitrogen limitation [25,99,103,118].…”

Section: Cultivation-level Strategiesmentioning

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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“…Many strategies could be developed in order to decrease proteolysis of secreted protein: pH modification (Werten et al, 1999;Scorer et al, 1993), complementation by elements rich in amino acids, such as casamino acids or peptone (Clare et al, 1991b), or use of protease-deficient host strains (Cregg et al, 1993). In this study, we examined the effects of pH modification and addition of casamino acids.…”

Section: Proteolysis and Ph Culture Effectmentioning

confidence: 99%

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Céline¹,

Chemardin²,

Bigey³

et al. 2004

Yeast

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Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae α-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theorical and practical purposes.

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High‐yield secretion of recombinant gelatins by Pichia pastoris

Cited by 82 publications

References 30 publications

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

Heterologous protein production using the Pichia pastoris expression system

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

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