Expression inPichia pastorisand Purification ofAspergillus awamoriGlucoamylase Catalytic Domain

Heimo, Heikki; Palmu, Kaisa; Suominen, Ilari

doi:10.1006/prep.1996.0713

Cited by 37 publications

(19 citation statements)

References 26 publications

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“…This organism has shown great potential in heterologous gene expression (12). Although several mammalian and bacterial proteins have been expressed in high levels in P. pastoris, relatively little has been reported about the expression of fungal proteins in this host (35)(36)(37)(38)(39). The S. cerevisiae invertase (SUC2) signal has been used to direct the secretion of foreign protein in P. pastoris (40 -43).…”

Section: Discussionmentioning

confidence: 99%

High-Level Production of Recombinant Fungal Endo-β-1,4-xylanase in the Methylotrophic Yeast Pichia pastoris

Berrin

Williamson

Puigserver³

et al. 2000

Protein Expression and Purification

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Section: Discussionmentioning

confidence: 99%

High-Level Production of Recombinant Fungal Endo-β-1,4-xylanase in the Methylotrophic Yeast Pichia pastoris

Berrin

Williamson

Puigserver³

et al. 2000

Protein Expression and Purification

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“…The extent of O-glycosylation by P. pastoris has been studied in a glucoamylase catalytic domain from Aspergillus awamori [44]. The molecular weight of the secreted protein was 20 kDa heavier than the native protein.…”

Section: O-and N-linked Glycosylationmentioning

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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“…FPLC-purified recombinant chitinase BjCHI1, BjCHI2 or BjCHI3 (3 lg) was incubated for 6 days at 30°C with 1 unit of a-mannosidase in a 30-ll solution of 20 mM sodium acetate, pH 4.5, and 1 mM zinc sulfate (Heimo et al, 1997). In each control sample, the chitinase without amannosidase was similarly treated.…”

Section: Deglycosylation Of Recombinant Chitinases Bjchi1 Bjchi2 Andmentioning

confidence: 99%

Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

et al. 2004

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We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using alpha-mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by alpha-mannosidase. BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity. H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis. E234A showed 36% retention of activity and substitution Y269D, 50%. The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.

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Expression inPichia pastorisand Purification ofAspergillus awamoriGlucoamylase Catalytic Domain

Cited by 37 publications

References 26 publications

High-Level Production of Recombinant Fungal Endo-β-1,4-xylanase in the Methylotrophic Yeast Pichia pastoris

High-Level Production of Recombinant Fungal Endo-β-1,4-xylanase in the Methylotrophic Yeast Pichia pastoris

Heterologous protein production using the Pichia pastoris expression system

Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

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