Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry

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Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry. / Dionisio, Giuseppe; Jørgensen, Malene; Welinder, Karen Gjesing; Brinch-Pedersen, Henrik.

I: Protein Expression and Purification, Bind 82, Nr. 1, 03.2012, s. 179-185.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Dionisio, Giuseppe ; Jørgensen, Malene ; Welinder, Karen Gjesing ; Brinch-Pedersen, Henrik. / Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry. I: Protein Expression and Purification. 2012 ; Bind 82, Nr. 1. s. 179-185.

Bibtex

@article{664a6a054284453e852f129054867dab,
title = "Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry",
abstract = "Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.",
keywords = "Mass spectrometry of hyper glycosylated peptides, Pichia pastoris secreted protein, Posttranslational modification in Pichia, Recombinant phytase, Wheat phytase",
author = "Giuseppe Dionisio and Malene J{\o}rgensen and Welinder, {Karen Gjesing} and Henrik Brinch-Pedersen",
year = "2012",
month = mar,
doi = "10.1016/j.pep.2011.12.003",
language = "English",
volume = "82",
pages = "179--185",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",
number = "1",

}

RIS

TY - JOUR

T1 - Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry

AU - Dionisio, Giuseppe

AU - Jørgensen, Malene

AU - Welinder, Karen Gjesing

AU - Brinch-Pedersen, Henrik

PY - 2012/3

Y1 - 2012/3

N2 - Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.

AB - Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.

KW - Mass spectrometry of hyper glycosylated peptides

KW - Pichia pastoris secreted protein

KW - Posttranslational modification in Pichia

KW - Recombinant phytase

KW - Wheat phytase

U2 - 10.1016/j.pep.2011.12.003

DO - 10.1016/j.pep.2011.12.003

M3 - Journal article

C2 - 22240269

VL - 82

SP - 179

EP - 185

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -