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2007 Fiscal Year Final Research Report Summary

Synthesis of large glycoprotein by use of chemical and expression methods

Research Project

Project/Area Number 18550155
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Chemistry related to living body
Research InstitutionYokohama City University

Principal Investigator

KAJIHARA Yasuhfro  Yokohama City University, 国際総合科学研究科, Professor (50275020)

Project Period (FY) 2006 – 2007
Keywordsoligosaccharide / glvcopeptide / disulfide bond / erythropoietine / glvcoprotein
Research Abstract

To investigate function of oligosaccharide on protein, we have examined synthesis of homogeneous glycoproteins by use of combined chemical and E. coli expression system. In our strategy, a homogeneous glycopeptide thioester is synthesized by chemical method and then coupling between this glycopeptide thioester and a large polypeptide chain prepared by E. coli expression system, is performed by Native Chemical Ligation (NCL). In this study, we finally selected erythropoietin (EPO) analogue, which consist of 166-amino acid residues and display two N-linked complex type disialyloligosaccharide chains at the 24 and 30 positions on the polypeptide chain. EPO analogue was divided into two segments, EPO segment (1-32) and EPO segment (33-166). In order to synthesize glycopeptides thioester segment having two cysteine residues at the 24 and 30 positions where would be incorporated with oligosaccharides, protected peptide chain (1-32) was prepared by Fmoc SPPS and then its C-terminal carboxylic acid was converted into ethyl-thioester to afford peptide thioester. A complex-type disialyloligosaccharyl-bromoacetamide was employed to modify the two free cysteine residues to obtain the neoglycopeptide thioester. This coupling reaction afforded the desired neoglycopeptide thioester (EPO:1-32) in good yield. The EPO polypeptide chain, comprising residues 33-166 was purified from E. coli as a polyhistidine-tagged fusion protein with a methionine residue inserted between the tag and the EPO sequence. After isolation of the fusion protein through nickel-affinity column, Histag-methionine segment was removed by CNBr treatment to afford desired EPO segment having cysteine residue at the N-terminus. Finally, we examined coupling reaction between these two segments by NCL and this reaction afforded whole EPO analogue in moderate yield. After folding experiment, we obtained desired EPO analogue by use of combined chemical and E. coil expression system.

  • Research Products

    (8 results)

All 2008 2007 2006

All Journal Article (2 results) (of which Peer Reviewed: 1 results) Presentation (5 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Convenient synthesis of a sialylglycopeptide-thioester having an intactand homogeneous complex-type disialyl-oligosaccharide2006

    • Author(s)
      Y. Kajihara, A. Yoshihara, K. Hirano, N. Yamamoto
    • Journal Title

      Carbohydrate Research 341

      Pages: 1333-1340

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Convenient synthesis of a sialylglycopeptide-thioester having an intact and homogeneous complex-type disialyI-oligosaccharide2006

    • Author(s)
      Y. Kajihara, A. Yoshihara, K. Hirano, N. Yamamoto
    • Journal Title

      Carbohydrate Research 341

      Pages: 11333-1340

    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] 化学法と発現法を組み合わせた糖タンパク質誘導体の合成研究2008

    • Author(s)
      平野 桐子、Derek Macmman、梶原 康宏
    • Organizer
      日本化学会第88春季年会
    • Place of Presentation
      立教大学(池袋キャンパス)
    • Year and Date
      20080326-30
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Chemical synthesis of glycoproteins having human complex type oligosac charide2007

    • Author(s)
      Yasuhiro Kajihara
    • Organizer
      4th International Peptide Synposium
    • Place of Presentation
      Cairns, Australia
    • Year and Date
      20071021-25
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Chemical synthesis of glycoproteins having human complex type oligosaccharides2007

    • Author(s)
      Yasuhiro, Kajihara
    • Organizer
      4th International Peptide Synposium
    • Place of Presentation
      Cairns, Australia
    • Year and Date
      20071021-25
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Chemical synthesis of glycoproteins having intact and homogeneous complex type oligosaccharides2007

    • Author(s)
      Yasuhiro Kajihara
    • Organizer
      Gordon Reserch Conferences CARBOHYDRATES
    • Place of Presentation
      Tilton School Tilton,USA
    • Year and Date
      20070617-22
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Chemical synthesis of glycoproteins having intact and homogeneous complex type oligosaccharides2007

    • Author(s)
      Yasuhiro, Kajihara
    • Organizer
      Gordon Reserch Conferences
    • Place of Presentation
      Tilton, USA
    • Year and Date
      20070617-22
    • Description
      「研究成果報告書概要(欧文)」より
  • [Patent(Industrial Property Rights)] 均一な糖鎖構造を有するエリスロポエチン誘導体2008

    • Inventor(s)
      梶原 康宏, 他4名
    • Industrial Property Rights Holder
      横浜市立大学 大塚化学
    • Industrial Property Number
      特願 2008-61913
    • Filing Date
      2008-03-11
    • Description
      「研究成果報告書概要(和文)」より

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Published: 2010-02-04  

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