Structural and functional characterization of recombinant human serum transferrin secreted from Pichia pastoris.
Serum transferrin is an iron-binding glycoprotein with a bilobal structure. It binds iron ions in the blood serum and delivers them into target cells via transferrin receptor. We identified structural and functional characteristics of recombinant human transferrin which is produced in the yeast Pichia pastoris. Using the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae, high-level secretion was obtained, up to 30 mg/l of culture medium. Correct processing at designed sites was confirmed by N-terminal sequence analysis. Carbohydrate modification was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis after digestion with endo-beta-N-acetylglucosaminidase H. Reflecting the secondary structure, the circular dichroism spectrum of the recombinant protein was indistinguishable from that of serum transferrin. Consequently, the recombinant product had an iron binding function just as the serum specimen has: two Fe(3+) sites existed in a recombinant transferrin molecule, as estimated by titration analysis using visible absorption, fluorescence spectra, and electrophoretic behavior in urea denaturing polyacrylamide gel electrophoresis (PAGE).
Graduate School of Agriculture, Kyoto University, Uji, Japan. kmizutani@kais.kyoto-u.ac.jp
Kimihiko
K
Mizutani
Kazuhiko
K
Hashimoto
Nobuyuki
N
Takahashi
Masaaki
M
Hirose
Shigeo
S
Aibara
Bunzo
B
Mikami
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014168:Transferrin / Q000235:genetics / Q000378:metabolism*
D002241:Carbohydrates; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron