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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^†^,^‡

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,^§ and Ross T. A. MacGillivray*

Institute of Molecular Biosciences, College of Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand, Departments of Biochemistry and Molecular Biology and of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, and Department of Biochemistry, University of Vermont, Burlington, Vermont 05405-0068

Biochemistry, 1999, 38 (8), pp 2535–2541

DOI: 10.1021/bi9824543

Publication Date (Web): February 2, 1999

†

Supported by grants from the U. S. Public Health Service (R01 DK 21739 to R.C.W. and R01 HD 20859 to E.N.B.), and the Medical Research Council of Canada (to R.T.A.M.). During his stay at Massey University, R.T.A.M. was supported in part by a Killam Senior Research Fellowship and by USPHS Grant R01 DK 35533. E.N.B. also acknowledges research support as an International Research Scholar of the Howard Hughes Medical Institute.

‡

Atomic coordinates of the protein structure reported in this paper has been deposited with the Protein Data Bank at Brookhaven National Laboratories with PDB Code 1b3e.

Massey University.

Current address: Brookhaven National Laboratory, Upton, NY 11973-5000.

Department of Biochemistry and Molecular Biology, University of British Columbia.

Department of Chemistry, University of British Columbia.

Current address: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

University of Vermont.

Corresponding author. Tel: (604)822.3027. Fax: (604)822.4364. E-mail: macg@unixg.ubc.ca.

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.

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