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Random Insertion of Green Fluorescent Protein into the Regulatory Subunit of Cyclic Adenosine Monophosphate-Dependent Protein Kinase

  • Pascal J. Baehler
  • Ricardo M. Biondi
  • Miguel van Bemmelen
  • Michel Véron
  • Christophe D. Reymond
Part of the Methods in Molecular Biology book series (MIMB, volume 183)

Abstract

Various fusion proteins have been made with green fluorescent proteins (GFPs) (for a recent review, see ref. 1) as a means to localize such proteins within living cells (2). In most cases, GFPs were added at either the C- or Nterminal end of the protein or polypeptide of interest (3). For certain purposes, sueh as fluorescence resonance energy transfer (FRET), GFPs need to be placed at particular locations within the protein (4). Because the crystal structures of most proteins and protein complexes are not currently known, it is not usually possible to predict the optimal position for insertion of the GFP to obtain FRET, or to retain target protein activity. We thus devised a method for random insertion of GFPs within a target protein. The method generates a collection of fusion proteins that can be tested for a desired function.

Keywords

Fusion Protein Fluorescence Resonance Energy Transfer Wash Buffer Isoamyl Alcohol Linear Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Pascal J. Baehler
    • 1
  • Ricardo M. Biondi
    • 2
  • Miguel van Bemmelen
    • 3
  • Michel Véron
    • 3
  • Christophe D. Reymond
    • 1
  1. 1.Institut de Biologie Cellulaire et de MorphologieLausanneSwitzerland
  2. 2.Division of Signal Transduction TherapyUniversity of DundeeDundeeUK
  3. 3.Unité de Régulation Enzimatique des Activités CellulaireInstitut PasteurParisFrance

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