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Examination of Three Alternative Devices to Refold Correctly the Denatured Enzyme Originated from the Precipitate Produced in the Process of Protein Synthesis

Research Project

Project/Area Number 03555180
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 有機工業化学
Research InstitutionNagoya City University

Principal Investigator

SAKAI Tomoya  Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemical Reaction Engineering, Professor, 薬学部, 教授 (00080169)

Co-Investigator(Kenkyū-buntansha) FUKUHARA Kenichi  Ajinomoto, Co.Ltd., Central Res.Lab., Senior Researcher, 中央研究所, 主任研究員
KURIMOTO Eiji  Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 助手 (90234575)
KURODA Yoshitaka  Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 講師 (40080204)
NOHARA Daisuke  Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 助教授 (60080214)
Project Period (FY) 1991 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥800,000 (Direct Cost: ¥800,000)
KeywordsSubtilisin / Ribonuclease A / Lysozyme / Guanidine hydrochloride / Refolding / Globular protein / Recombinant DNA / Immobilization / Denaturant / タンパク質の折りたたみ / タンパク質の構造 / タンパク質の沈殿 / タンパク質の固定化 / タンパク質の不溶化 / 疎水結合からなるコアの形成 / リゾチ-ム / タンパク質のリフォ-ルディング / タンパク質の三次元構造 / カルボキシ末端固定 / アミノ末端固定 / 疎水性コア形成
Research Abstract

Protein refolding is one of the critical processes in the downstream of the recombinant DNA protein synthesis. The objective of the present research work is to evaluate comparatively following three devices for the correct refolding of a globular protein from its random coil structure produced, for example, in 6M GdnHCl ; that is, (1) to build the hydrophobic core in the first place, (2) to let it refold hopefully from its carboxy-terminal by immobilization at the amino-terminal, (3) to let it refold hopefully from its amino-terminal by immobilization at the carboxy-terminal.
Experimentally, hydrophobic core formation in the first place, (1), was realized by use of a refolding medium of high ionic strength ; immobilization of either N or C-terminal, (2) or (3), was conducted by means of covalently bonding on sepharose gel beads according to the known methods.
Of course, such irreversible covalent fixation of the protein might not meet practical uses, in protein refolding, however, it was … More adopted to get more precise evaluation for the above-mentioned three devices. Adopted globular proteins were bovine pancreatic ribonuclease A(RNase), hen egg-white lysozyme(Lyzm), and bacterial subtilisin BPN'(Sbtl).
It became clear in the course of examination that RNase and Lyzm were not suitable for the present test. RNase could not be immobilized at C-terminal retaining active form due to intramolecular amidation and immobilized Lyzm was not tolerable for the generalized activity assay procedure which uses cell walls as a substrate. Fortunately, by use of Sbtl, it was possible to examine exactly the above-mentioned three devices for the correct folding.
As for the device (1) which intends to form by hydrophobic core at first, we succeeded to refold 6M GdnHCl-denatured Sbtl at pH 2.4 by incubating it in 1.5-2.0M K-acetate at pH 6.5. However, the protease Sbtl suffers from autoproteolytic digestion to get a resultant maximum refolding yield of up to 30%. A precise evaluation of this device was attained by deletion of an autolysis event through the immobilization as follows.
Device (2) or (3) which corresponds to the refolding of Sbtl with N-terminal or C-terminal immobilized, respectively, was conducted by repeated dissolution of immobilized Sbtl in 6M GdnHCl followed by its refolding in 2M K-acetate. In both cases, almost 100% refolding yield was achieved after the 3rd denaturation/renaturation cycle based on the recovered activity of the preceding cycle.
Rate of refolding in the case of device (2) appeared to be larger than that of device (3) Moreover, a refolding medium consisted of 2M K-acetate was superior to that of 2M KCl. In conclusion, almost quantitative refolding of Sbtl was achieved in all three cases, provided that a suitable refolding environment was given. Among them, the most important device might be a hydrophobic core formation in the refolding of Sbtl followed by N-terminal and C-terminal immobilization. Less

Report

(4 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] M.Matsubara,D.Nohara,T.Sakai: "Difference Between Guanidinium Chloride and Urea as Denaturants of Globular Proteins:The Possibility of Application to Improved Refolding Processes" Chemical & Pharmaceutical Bulletin. 40. 550-552 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara,D.Nohara,E.Kurimoto,Y.Kuroda,T.Sakai: "“Loose Folding"and“Dlayed Oxidation"Procedures Successfully Applied for Refolding of Fully Reduced Hen Egg-White Lysozyme" Chemical & Pharmaceutical Bulletin. 41. 1207-1210 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara,E.Kurimoto,S.Kojima,K.Miura,T.Sakai: "Quantitative in vitro Renaturation of Subtilisin BPN' without the Aid of Pro-sequence" CHEMISTRY LETTERS. 1783-1786 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Hayashi,M.Matsubara,E.Kurimoto,D.Nohara,T.Sakai: "Refolding of Subtilisin BPN' Achieved Almost Quantitatively by Covalent Immobilization on an Agarose Gel" Chemical & Pharmaceutical Bulletin. 41. 2063-2065 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara,E.Kurimoto,S.Kojima,K.Miura,T.Sakai: "Achievement of in vitro Renaturation of Subtilisin BPN' by a Novel Procedure Using Organic Salts and a Digestible Mutant of Streptomyces Subtilisin Inhibitor" FEBS Letters. (in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara, D.Nohara, T.Sakai: "Difference Between Guanidinium Chloride and Urea as Denaturants of Globular Proteins : The Possibility of Application to Improved Refolding Processes" Chemical & Pharmaceutical Bulletin. Vol.40. 550-552 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara, D.Nohara, E.Kurimoto, Y.Kuroda, T.Sakai: ""Loose Folding" and "Delayd Oxidation" Procedures Successfully Applied for Refolding of Fully Reduced Hen Egg-White Lysozyme" Chemical & Pharmaceutical Bulletin. Vol.41. 1207-1210 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara, E.Kurimoto, S.Kojima, K.Miura, T.Sakai: "Quantitative in vitro Renaturation of Subtilisin BPN' without the Aide of Pro-sequence" CHEMISTRY LETTERS. 1783-1786 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Hayashi, M.Matsubara, E.Kurimoto, D.Nohara, T.Sakai: "Refolding of Subtilisin BPN' Achieved Almost Quantitatively by Covalent Immobilization on an Agarose Gel" Chemical & Pharmaceutical Bulletin. Vol.41. 2063-2065 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara, E.Kurimoto, S.Kojima, K.Miura, T.Sakai: "Achievement of in vitro Renaturation of Subtilisin BPN' by a Novel Procedure Using Organic Salts and a Digestible Mutant of Streptomyces Subtilisin Inhibitor" FEBS Letters. (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Matsubara,D.Nohara,T.Sakai: "Difference Between Guanidinium Chloride and Urea as Denaturants of Globular Proteins:The Possibility of Application to Improved Refolding Processes" Chemical & Pharmaceutical Bulletin. 40. 550-552 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Matsubara,D.Nohara,E.Kurimoto,Y.Kuroda,T.Sakai: "“Loose Folding"and“Delayed Oxidation"Procedures Successfully Applied for Refolding of Fully Reduced Hen Egg-White Lysozyme" Chemical & Pharmaceutical Bulletin. 41. 1207-1210 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Matsubara,E.Kurimoto,S.Kojima,K.Miura,T.Sakai: "Quantitative in vitro Renaturation of Subtilisin BPN' without the Aid of Pro-seguence" CHEMISTRY LETTERS. 1783-1786 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Hayashi,M.Mathubara,E,Kurimoto,K.Nohara,T.Sakai: "Refolding of Subtilisin BPN' Achieved Almost Quantitatively by Covalent Immobilization on an Agarose Gel" Chemical & Pharmaceutical Bulletin. 41. 2063-2065 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Mathubara,E.Kurimoto,S.Kojima,K.Miura,T.Sakai: "Achievement of in vitro Renaturation of Subtilisin BPN' by a Novel Procedure Using Organic Salts and a Digestible Mutant of Streptomyces Subtilisin Inhibitor" FEBS Letters. (in press). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Matsubara: "¨Loose Folding¨and¨Delayed Oxidation¨Procedures Successfully Applied for Refolding of Fully Reduced Hen Egg White Lysozyme" Chem.Pharm.Bull.(1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Matsubara: "Difference between Guanidinium Chloride and Urea as Denaturants of Globular Proteins:The Possibility of Application to Improved Refolding Processes" Chem.Pharm.Bull.40(2). 550-552 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Mamoru MATSUBARA: "Difference between guanidinium chlovide and urea as denaturants of globular proteins:The possibility of application to improved refulding processes." Chemical & Pharmaceutical Bulletin. 40. 550-552 (1992)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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