Detection of protein-protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer

Seiji Shibasaki, Kouichi Kuroda, Duc Nguyen Hoang, Tomoaki Mori, Wen Zou, Mitsuyoshi Ueda

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)-protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein-protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP-protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP-protein A and EYFP-Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein-protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei.

Original languageEnglish
Pages (from-to)451-457
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume70
Issue number4
DOIs
Publication statusPublished - Apr 2006
Externally publishedYes

Fingerprint

Recombinant proteins
Fluorescence Resonance Energy Transfer
Recombinant Proteins
Cell Membrane
Proteins
Membranes
Cell membranes
Staphylococcal Protein A
Protein Transport
Immunoglobulin Fc Fragments
Two-Hybrid System Techniques
Biotechnology
Molecular interactions
Saccharomyces cerevisiae
Cytoplasm
Yeast
Immunoglobulin G
Fluorescence
Fusion reactions
Display devices

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

Detection of protein-protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer. / Shibasaki, Seiji; Kuroda, Kouichi; Hoang, Duc Nguyen; Mori, Tomoaki; Zou, Wen; Ueda, Mitsuyoshi.

In: Applied Microbiology and Biotechnology, Vol. 70, No. 4, 04.2006, p. 451-457.

Research output: Contribution to journalArticle

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