Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 74, Issue 2, Pages 309-315Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.90635
Keywords
transferrin; iron transport; Pichia pastoris; carbohydrate chain
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Funding
- Japan Society for the Promotion of Science
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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 Fe3+ 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).
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