Preparation of Functional Single-Chain Antibodies against Bioactive Gibberellins by Utilizing Randomly Mutagenized Phage-Display Libraries

• SUZUKI Yoshihito

  Department of Applied Biological Chemistry, The University of Tokyo

• ITO Shinsaku

  Department of Applied Biological Chemistry, The University of Tokyo

• OTSUKA Kaori

  Department of Applied Biological Chemistry, The University of Tokyo

• IWASAWA Eriko

  Department of Applied Biological Chemistry, The University of Tokyo

• NAKAJIMA Masatoshi

  Department of Applied Biological Chemistry, The University of Tokyo

• YAMAGUCHI Isomaro

  Department of Applied Biological Chemistry, The University of Tokyo

抄録

Screening randomly mutagenized proteins displayed on a phage surface by biopanning is a powerful strategy to obtain evolved clones with improved properties such as higher stability and functionality. We utilized this method to overcome the problem that functional single-chain antibodies against active gibberellins, a class of plant hormones, can not be prepared by some of the conventional methods. Single-chain antibody libraries with random mutations were constructed from two independent anti-bioactive gibberellin monoclonal antibody lines in a phagemid vector, so that the mutagenized scFvs were expressed in a phage-displayed form upon helper phage infection. From both libraries, scFv clones with binding activity to GA₄ were successfully obtained by successive rounds of biopanning against BSA-GA₄, the original immunogen. The results are highly suggestive that this approach might be a general solution when a single-chain antibody does not show binding activity. We found further that a ribosomal frameshift to complement a nonsense mutation frequently occurred in an amber suppressor strain of E. coli TG1, resulting in the display of a functional antibody, while such a nonsense mutant failed to produce a soluble antibody in a non-amber suppressor strain. This result explains at least partly why single-chain antibodies are sometimes functional only in a phage-displayed form, not in a soluble form.

収録刊行物

• Ｂｉｏｓｃｉｅｎｃｅ，　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，　ａｎｄ　Ｂｉｏｃｈｅｍｉｓｔｒｙ

  Ｂｉｏｓｃｉｅｎｃｅ，　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，　ａｎｄ　Ｂｉｏｃｈｅｍｉｓｔｒｙ 69 (3), 610-619, 2005

  公益社団法人 日本農芸化学会

キーワード

• single-chain Fv
• gibberellin
• phage-display library
• random mutagenesis

詳細情報

• CRID: 1390001206473996928
• NII論文ID: 130000030401
• NII書誌ID: AA10824164
• DOI: 10.1271/bbb.69.610
• ISSN: 13476947
• ISSN: 09168451
• NDL書誌ID: 7295584
• PubMed: 15784991
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I7295584
• Web Site: http://www.tandfonline.com/doi/pdf/10.1271/bbb.69.610
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可