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. Journal   Biochemistry. Citation   Biochemistry. 38(8):2535-41 Publication date   1999 Feb 23 Authors   Bewley MC Tam BM Grewal J He S Shewry S Murphy ME Mason AB Woodworth RC Baker EN MacGillivray RT Investigators   Maria C. Bewley Grant agencies   National Institute of Diabetes and Digestive and Kidney Diseases National Institute of Child Health and Human Development Grants   NIDDK R01 DK 21739 NIDDK R01 DK 35533 NICHD R01 HD 20859 MeSH headings   Peptide Fragments Pichia Protein Folding Recombinant Proteins Serine Transferrin MeSH qualifiers   chemistry genetics metabolism 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. Medline ID   99155227