Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

İnan, Mehmet; Aryasomayajula, Dinesh; Sinha, Jai B. P.; Meagher, Michael M.

doi:10.1002/bit.20762

Cited by 158 publications

(98 citation statements)

References 27 publications

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“…It is reported that overexpression of protein disulfide isomerase could enhance protein secretion in Pichia pastoris (Inan et al 2006). Considerable deviations in the secondary structure of rCel7A compared to H. jecorina Cel7A, revealed by circular dichroism, were also observed in previous studies (Godbole et al 1999;Boer et al 2000).…”

Section: Discussionmentioning

confidence: 71%

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

Wei

Liu

et al. 2009

World J Microbiol Biotechnol

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In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.

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Section: Discussionmentioning

confidence: 71%

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

Wei

Liu

et al. 2009

World J Microbiol Biotechnol

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“…Optimization of Heterologous Expression-To overcome the low level of expression for BBE H104A and H104A-C166A reported previously (20), we attempted to improve the level of secretory expression in Pichia pastoris by co-overexpression of protein disulfide isomerase (PDI), a protein reported to have a general positive effect on the secretory machinery in yeasts (22,23). The plasmid containing the coding sequence of PDI from P. pastoris in the vector pPICK was a generous gift of Prof. A. Glieder and Dr. S. Abad.…”

Section: Methodsmentioning

confidence: 99%

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Winkler

Motz

Riedl

et al. 2009

Journal of Biological Chemistry

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Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8␣-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8␣-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8␣-histidylation (either to N1 or to N3), 8␣-O-tyrosylation, 8␣-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX) 4 from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8␣-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7-9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12)...

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“…These auxiliary chaperones can load substrates into the HSP90 molecular chaperone [24], assist in the folding of heterologous proteins, and re-target substrates into degradable pathways [9]. Disulfide isomerase (PDI) is responsible for the catalytic acceleration of further folding of heterologous proteins [25]. PDI has two bioactivities: it acts as a disulfide bond-dependent and bond-independent molecular chaperone, and is responsible for the formation and isomerization of the disulfide bond [26].…”

Section: Endoplasmic Reticulum Resident Proteins and Their Effect On mentioning

confidence: 99%

Improving the Secretory Production of the Heterologous Protein in Pichia pastoris by Focusing on Protein Folding

Zhu

Chen

et al. 2014

Appl Biochem Biotechnol

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Pichia pastoris has currently been developed as an effective host system for the expression of heterologous genes owing to its potential use for the production of soluble and high-yield proteins. However, the secretory production of the different heterologous proteins in P. pastoris varies widely. Some factors restrict the effective secretory production of heterologous proteins in P. pastoris, among which the folding and processing of proteins is a major one. Besides optimizing the fermentative process, current strategies focus on investigating protein folding process. Thus, this paper is the first time to review the improvement of the secretory production of the heterologous protein in P. pastoris by focusing on its folding process.

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Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

Cited by 158 publications

References 27 publications

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Improving the Secretory Production of the Heterologous Protein in Pichia pastoris by Focusing on Protein Folding

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