Application of the green fluorescent protein as a reporter for Ace1-based, two-hybrid studies

Biotechniques. 1999 Jul;27(1):86-8, 92-4. doi: 10.2144/99271st01.

Abstract

The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo. This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex if interacting proteins are fused to these domains. Interactions are consequently detected through the activity of reporter genes. The two-hybrid technology has been successfully applied for the determination of interactions between numerous proteins of several organisms. Conventional reporter systems, such as the beta-galacatosidase from Escherichia coli, suffer from a variety of drawbacks, including the requirement for external substrates. In this report, we describe an alternative version of the two hybrid system using the combined advantages of the copper-inducible transcription factor Acel together with the yeast metallothionein gene CUP1 and the green fluorescence protein from aquatic invertebrates as reporters. This technique allows the copper-dependent monitoring of protein-protein interactions in living yeast cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins
  • Copper / pharmacology
  • DNA-Binding Proteins / genetics*
  • Genes, Reporter
  • Genetic Techniques*
  • Green Fluorescent Proteins
  • Luminescent Proteins*
  • Metallothionein / genetics
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins*
  • Transcription Factors / genetics*

Substances

  • CUP1-1 protein, S cerevisiae
  • CUP2 protein, S cerevisiae
  • Carrier Proteins
  • DNA-Binding Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • copper thionein
  • Green Fluorescent Proteins
  • Copper
  • Metallothionein