A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction

• 大室, 有紀

• Ohmuro-Matsuyama, Yuki

• 上田, 宏

• Ueda, Hiroshi

Abstract

Protein-fragment complementation assays (PCAs) are commonly used to assay protein–pro- tein interaction (PPI). While PCAs based on irely luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are diicult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we devel- oped a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more eicient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more sta- ble probes, and higher signal readout in a shorter time period, and it also worked in cellulo.

identifier:oai:t2r2.star.titech.ac.jp:50419883

Journal

• Protein-Protein Interaction Assays

  Protein-Protein Interaction Assays 11-27, 2018-07

  IntechOpen

Details

• CRID: 1050564288403426944
• NII Article ID: 120006582691
• DOI: 10.5772/intechopen.75644
• Web Site: http://t2r2.star.titech.ac.jp/cgi-bin/publicationinfo.cgi?q_publication_content_number=CTT100769945
• Web Site: http://www.intechopen.com/download/pdf/60569
• Text Lang: en
• Article Type: article
• Data Source:
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  • Crossref
  • CiNii Articles
  • KAKEN