A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing.
We describe a new method with general applicability for monitoring any protein-protein interaction in vivo. The principle is based on a protein splicing system, which involves a self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to formation of a new protein in which the exteins are linked directly by a peptide bond. As the exteins, split N- and C-terminal halves of enhanced green fluorescent protein (EGFP) were used. When a single peptide consisting of an intein derived from Saccharomyces cerevisiae intervening the split EGFP was expressed in Escherichia coli, the two external regions of EGFP were ligated, thereby forming the EGFP corresponding fluorophore. Genetic alteration of the intein, which involved large deletion of the central region encoding 104 amino acids, was performed. In the expression of the residual N- and C-terminal intein fragments each fused to the split EGFP exteins, the splicing in trans did not proceed. However, upon coexpression of calmodulin and its target peptide M13, each connected to the N- and C-terminal inteins, fluorescence of EGFP was observed. These results demonstrate that interaction of calmodulin and M13 triggers the refolding of intein, which induces the protein splicing, thereby folding the ligated extein correctly for yielding the EGFP fluorophore. This method opens a new way not only to screen protein-protein interactions but also to visualize the interaction in vivo in transgenic animals.
Ozawa
T
T
Department of Chemistry, School of Science, University of Tokyo, Japan.
Nogami
S
S
Sato
M
M
Ohya
Y
Y
Umezawa
Y
Y
D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008958:Models, Molecular; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D002147:Calmodulin; D008164:Luminescent Proteins; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009219:Myosin-Light-Chain Kinase