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

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  • Giuseppe Dionisio
  • Malene Jørgensen, Section for Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark
  • Karen Gjesing Welinder, Section for Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark
  • Henrik Brinch-Pedersen

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.

Original languageEnglish
JournalProtein Expression and Purification
Volume82
Issue1
Pages (from-to)179-185
Number of pages7
ISSN1046-5928
DOIs
Publication statusPublished - Mar 2012

    Research areas

  • Mass spectrometry of hyper glycosylated peptides, Pichia pastoris secreted protein, Posttranslational modification in Pichia, Recombinant phytase, Wheat phytase

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