Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.
In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.
Paulmurugan
R
R
The Crump Institute for Molecular Imaging, Los Angeles, California, USA.
Gambhir
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D005816:Genetic Complementation Test; D051798:Inhibitor of Differentiation Protein 1; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D051798:Inhibitor of Differentiation Protein 1; D008164:Luminescent Proteins; D017570:MyoD Protein; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors