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Analysis of the higher-order structure formation from the amino terminus of protein

Research Project

Project/Area Number 02680221
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionKobe University

Principal Investigator

TACHIBANA Hideki  Kobe Univ., Dept. Biology, Assoc. Prof., 理学部, 助教授 (70126118)

Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
KeywordsProtein / Module / Lysozyme / Renaturation / Folding / 構造形成
Research Abstract

A new direct expression vector, ATG-TAG vector, was developed, and applied to the expression in Escherichia coli of hen lysozyme module combinations 1+2+3+4+5(hereafter abbreviated as Ml-5, and so on), Ml-4, Ml-3, M2-5 and M2-4. The expression of Mi-2 and Ml as fused proteins was not carried out. The repression of expression during uninduced period was observed when minimal media were used, but was not when rich media were used. Variation in IPTG concentration as well as in the culture temperature so far used did not lead to the expression into a soluble fraction. The primary structures of the expressed materials were as expected. These module combinations, when disulfide-bridges were opened, showed the CD spectra which indicated a decrease in alpha-helix and an increase in beta-sheet contents compared to authentic native lysozyme. This 'residual' structure showed a non-cooperative transition on GuHCl-induced denaturation. Glycerol, sorbitol, methanol and trifluoroethanol increased the … More secondary structure of the reduced module combinations. Renatured(reoxidized)module combinations contained, ' except for Ml-5 and M2-4, more than ten molecular species of different higher-order structure as monitored by RPHPLC. M2-5 showed an increase in higher-order structure when reoxidized in the presence of polyols or alcohols. Cooperative formation of tertiary structure, however, was not observed. on the other hand, the lysozyme derivatives which lacked one disulfide bond while retaining fulllength polypeptide were renatured efficiently in the presence of glycerol, and showed lytic activities and secondary structures comparable to those of authentic lysozyme. Another derivative which contained residues 21 to 129, and which could form three native disulfide bonds, did not show a 'correct' folding even in the presence of glycerol. Altogether, both amino-terminal(residues 1 to 20)and carboxyl-terminal region(108-129)are necessary for the correct higherorder structure formation of hen lysozyme. Less

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Tachibana,H.: "An 'initiator-terminator vector' suitable for direct expression of genic segments in Escherichia coil." Protein Engineering. 3. 371 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sato,K.: "A synthetic peptide corresponding to residues 137 to 157 of p60^<v-src> inhibits tyrosine-epecific protein kinases." Biochemical and Biopyhsical Res.Communications. 171. 1152-1159 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tachibana,H.: "Construction of an 'ATG-TAG vector' and direct expression of chicken lysozyme structural domains in E.coli." Gene.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sawano,H.: "Efficient in vitro folding of the three-disulfide derivatives of hen lysozyme in the presence of glycerol." FEBS Lerrers.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tachibana, H., Sasaki, Y., Sawano, H. and Kitakawa, M.: "An 'initiator-terminator vector' suitable for direct expression of genic segments" Escherichia coli. Protein Engineering. 3. 371 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T. and Fukami, Y.: "A synthetic peptide corresponding to residues 137 to 157 of p60^<v-src> inhibits tyrosine-specific protein kinases." Biochemical and Biophysical Research Communications. 171. 1152-1159 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tachibana, H., Sasaki, Y., Sawano, H., Koumoto, Y., Ohta, K. and Kitakawa, M.: "Construction of an 'ATG-TAG vector' and direct expression of chicken lysozyme structural domains in Escherichia coli."

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sawano, H., Koumoto, Y., Ohta, K., Sasaki, Y., Segawa, S. and Tachibana, H.: "Efficient in vitro folding and disulfide bond formation of the three-disulfide derivatives of hen lysozyme in the presence of glycerol."

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 澤野 博志: "ニワトリリゾチ-ム・ジスルフィド結合欠損体の作製" 生物物理. 31. S227 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 大田 勝之: "ニワトリリゾチ-ムモジュ-ル結合体再生物の構造と酵素活性" 生物物理. 31. S227 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Tachibana,H.: "Construction of an‘ATCーTAG vector'and direct expression of chicken lysozyme Structural domains in Escherichia coli." Gene.

    • Related Report
      1991 Annual Research Report
  • [Publications] Sawano,H.: "Efficient in vitro folding of the threeーdisulfife derivatives of hen lysozyme in the presence of glycerol." FEBS Letters.

    • Related Report
      1991 Annual Research Report
  • [Publications] Tachibana,H.: "An'initiatorーterminator vector' suitable for direct expression of genic segmens in Escherichia coli" Protein Engineering. 3. 371 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 大田 勝之: "カルボキサミドメチル化リゾチ-ムモジュ-ル複合体の残存構造" 生物物理. 30. S319 (1990)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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