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 Pich...
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Published in | Bioscience, biotechnology, and biochemistry Vol. 74; no. 2; pp. 309 - 315 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Tokyo
Japan Society for Bioscience, Biotechnology, and Agrochemistry
2010
Japan Society for Bioscience Biotechnology and Agrochemistry Oxford University Press |
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Abstract | 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 α 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-β-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). |
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AbstractList | 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 α 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-β-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). 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-b-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). 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). 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-b-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 super(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). 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 α 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-β-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). |
Author | Mizutani, K., Kyoto Univ., Uji (Japan) Takahashi, N Mikami, B Hashimoto, K Aibara, S Hirose, M |
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Cites_doi | 10.1021/bi00173a035 10.1016/S1046-5928(02)00528-4 10.1146/annurev.bi.49.070180.002041 10.1021/bi00415a061 10.1021/bi00083a016 10.1038/344784a0 10.1006/jmbi.2001.4719 10.1016/0003-2697(81)90281-5 10.1006/prep.2001.1607 10.1016/j.pep.2004.04.013 10.1271/bbb.61.2125 10.1006/jmbi.1995.0611 10.1042/BA20000069 10.1271/bbb.68.376 10.1107/S0907444900005151 10.1016/S0021-9258(19)84787-7 10.1107/S0907444996000212 10.1038/227680a0 10.1073/pnas.80.8.2263 10.1074/jbc.M102590200 10.1021/bi992574q 10.1107/S0907444999009865 10.1074/jbc.M604592200 10.1016/S0021-9258(18)54426-4 10.3390/i7070197 10.1107/S0907444901017309 10.1006/prep.1995.1081 10.1074/jbc.275.17.12463 10.1124/pr.54.4.561 10.1016/S0969-2126(94)00103-0 10.1021/bc800126s 10.1074/jbc.274.15.10190 |
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Keywords | Ascomycota Fungi Characterization Human Transferrin Serum Iron Carbohydrate Pichia pastoris Transport iron transport carbohydrate chain |
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SubjectTerms | Amino Acid Sequence Binding Sites - genetics Biological and medical sciences BIOTECHNOLOGIE BIOTECHNOLOGY BIOTECNOLOGIA BLOOD SERUM carbohydrate chain Carbohydrates - chemistry Fundamental and applied biological sciences. Psychology http://www.fao.org/aos/agrovoc#c_14958 http://www.fao.org/aos/agrovoc#c_16165 http://www.fao.org/aos/agrovoc#c_36920 http://www.fao.org/aos/agrovoc#c_37093 http://www.fao.org/aos/agrovoc#c_9323 Humans Iron - metabolism iron transport Molecular Sequence Data Molecular Weight Pichia - genetics Pichia - metabolism PICHIA PASTORIS Protein Binding Protein Structure, Secondary - genetics PROTEINAS RECOMBINANTES PROTEINE RECOMBINANTE RECOMBINANT PROTEINS Recombinant Proteins - chemistry Recombinant Proteins - metabolism Saccharomyces cerevisiae SERUM SANGUIN SUERO SANGUINEO transferrin Transferrin - genetics Transferrin - metabolism TRANSFERRINAS TRANSFERRINE TRANSFERRINS |
Title | Structural and functional characterization of recombinant human serum transferrin secreted from Pichia pastoris |
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