X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32

Maria C. Bewley, Beatrice M. Tam, Jasmine Grewal, Shouming He, Steven Shewry, Michael E.P. Murphy, Anne B. Mason, Robert C. Woodworth, Edward N. Baker, Ross T.A. MacGillivray

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

The ferric form of the N-lobe of human serum transferrin (Fe(III)- hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)- hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 Å resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris- derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.

Original languageEnglish (US)
Pages (from-to)2535-2541
Number of pages7
JournalBiochemistry
Volume38
Issue number8
DOIs
StatePublished - Feb 23 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry

Fingerprint Dive into the research topics of 'X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32'. Together they form a unique fingerprint.

Cite this