Protein Splicing-Based Reconstitution of Split Green Fluorescent Protein for Monitoring Protein−Protein Interactions in Bacteria:  Improved Sensitivity and Reduced Screening Time

Abstract

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.

Bioluminescent Indicators for Ca2+ Based on Split Renilla Luciferase Complementation in Living Cells

“…CaM, C124A, hRLn and hRLc were prepared as described previously. [9][10][11][12] Cell culture and transfection MCF-7 cells were cultured in MEM supplemented with 10% heat-inactivated fatal calf serum, 1 mM sodium pyruvate, 100 mM MEM non-essential amino-acid solution, 100 unit/mL penicillin and 100 mg/mL streptomycin. Cells were maintained in 5% CO2 at 37˚C.…”

Methods of Analysis for Imaging and Detecting Ions and Molecules

“…We have developed genetically encoded intracellular fluorescent and bioluminescent optical probes, which include second messengers, such as guanosine 3 0 ,5 0 -cyclic monophosphate (cGMP), 1 inositol 1,4,5-trisphosphate (IP 3 ), 2 lipid second messengers, 3,4 and nitric oxide (NO), 5 protein phosphorylations, [6][7][8] protein-protein interactions, [9][10][11][12][13][14] protein localizations, [15][16][17][18][19] and nuclear receptor ligands, 20,21 for monitoring intracellular signaling. These probes are not only for fundamental biological studies, but also for assay and screening of possible pharmaceutical or toxic chemicals that inhibit or facilitate cellular signaling pathways.…”

“…To monitor protein-protein interactions (PPIs), a new method with general applicability was developed based on protein splicing. 9,10 In this process, an intervening protein sequence is excised, and the flanking protein fragments are spliced together. In this splicing system, the flanking pieces are the N-and C-terminal fragments of split GFPs.…”

Methods of Analysis for Chemicals that Promote/Disrupt Cellular Signaling

“…12). [41][42][43][44] Unlike earlier protein interaction assays, the split-GFP system involves the reconstitution of GFP, and does not require that the protein-protein interactions occur near the cell nucleus and reporter genes or that an enzyme substrate be present. This will 512 ANALYTICAL SCIENCES MAY 2002, VOL.…”