Biochemistry. 1999 Feb 23;38(8):2535-41.

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.

Bewley MC, Tam BM, Grewal J, He S, Shewry S, Murphy ME, Mason AB, Woodworth RC, Baker EN, MacGillivray RT.

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Institute of Molecular Biosciences, College of Sciences, Massey University, Palmerston North, New Zealand.

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 A 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.

PMID:: 10029548; [PubMed - indexed for MEDLINE]

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Structures reported by this article

Human Serum Transferrin, N-Terminal Lobe, Expressed In Pichia Pastoris

PDB: 1B3E

Source: Homo sapiens

Method: X-Ray Diffraction

Resolution: 2.5 Å

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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.

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