Detecting protein-protein interactions using Renilla luciferase fusion proteins.
We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.
Burbelo
Peter D
PD
Lombardi Cancer Center and the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20057, USA. burbelpd@georgetown.edu
Kisailus
Adam E
AE
Peck
Jeremy W
JW
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000201:enzymology*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002522:Chlorocebus aethiops; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D008156:Luciferases / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D008156:Luciferases; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein