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

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) h as been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P4(1)2(1)2, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 Angstrom reso lution. This represents only the second P. pastoris-derived protein structu re determined to date, and allows the comparison of the structures of recom binant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with ser um-derived transferrin. The polypeptide folding pattern is essentially iden tical in all of the three proteins. Mass spectroscopic analyses of P. pasto ris- 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 f rom the iron-binding site, glycosylation of Ser-32 should not affect the ir on-binding properties of hTF/2N expressed in P. pastoris, making this an ex cellent expression system for the production of hTF/2N.