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) 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.
Institute of Molecular Biosciences, College of Sciences, Massey University, Palmerston North, New Zealand.
M C
MC
Bewley
B M
BM
Tam
J
J
Grewal
S
S
He
S
S
Shewry
M E
ME
Murphy
A B
AB
Mason
R C
RC
Woodworth
E N
EN
Baker
R T
RT
MacGillivray
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003460:Crystallization; D018360:Crystallography, X-Ray; D005290:Ferric Compounds / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D013058:Mass Spectrometry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005290:Ferric Compounds; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014168:Transferrin; D012694:Serine