X-ray Crystallography and Mass Spectroscopy Reveal that the N-lobe of Human Transferrin Expressed inPichia pastorisIs Folded Correctly but Is Glycosylated on Serine-32^,

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.

Transferrin-Mediated Targeting of Bacteriophage HK97 Nanoparticles Into Tumor Cells

“…Tf is a glycosylated protein [37,38] and the carbohydrates present a versatile target for bioconjugation [39]. Multiple copies of Tf were attached to either C164 or C385 P-II with a bivalent linker that displays a maleimide functional group and a hydra-zide separated by a 20-Å hydrocarbon chain.…”

High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

“…Heterogeneity in the recombinant product exists only in the form of O-linked glycosylation. The two peaks (Figure 7B (ii)) corresponded to the predicted mass of the non-N-linked glycosylated transferrin and a form containing a single hexose, probably resulting from yeast O-linked mannosylation at serine-32 [29]. Functional testing by receptor-mediated iron uptake (Table 2) and cell culture supplementation experiments [30] indicated equivalence between the plasma-derived and rTf analogue.…”

Partial Characterization of the Human Serum Transferrin Epitope Reactive with the Monoclonal Antibody TRC-2

“…(25)(26)(27)(28) This may explain the lack of effect of TRC-2 (which shows no interspecies cross-reactivity) in Tf h -TfR interactions; yet the apparent location of TRC-2 specific epitope in the first of the three conserved regions (i.e., homology block A) (33,35) suggests a precaution in such a correlative approach.…”