LuMPIS: luciferase-based MBP-pull-down protein interaction screening system

Methods Mol Biol. 2012;815:265-75. doi: 10.1007/978-1-61779-424-7_20.

Abstract

Analyzing the putative interaction partners of an individual protein is one approach to elucidate its function. In the LuMPIS protocol, bait and prey proteins are expressed with N-terminal maltose binding protein (MBP)- and eGFP-luciferase (eGFP-luc) tags, respectively. Positive protein-protein interactions (PPIs) can be detected after pull-down of the MBP-tagged prey protein using amylose beads followed by the bioluminescence detection of the bound eGFP-luc-tagged bait protein. The LuMPIS technology offers the following advantages: the PPIs are detected in the mammalian cell context, the use of two long protein tags (i.e., MBP and eGFP-luc) increases the expression levels of genes whose gene expression levels are known to be frequently impaired, the use of amylose beads for pull-down is much more economic as compared to sepharose beads in combination with monoclonal antibodies and finally, the use of an eGFP-luc-tag enables the qualitative control of transfection efficiencies by fluorescence microscopy prior to starting the assay.

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cells, Cultured
  • Chromatography, Affinity
  • Cloning, Molecular
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / isolation & purification
  • Humans
  • Luciferases, Renilla / chemistry*
  • Luciferases, Renilla / isolation & purification
  • Maltose-Binding Proteins / chemistry
  • Maltose-Binding Proteins / isolation & purification
  • Polymerase Chain Reaction
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Transfection
  • Viral Proteins / chemistry

Substances

  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Luciferases, Renilla