An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

• Published in: Analytical biochemistry Vol. 395; no. 2; pp. 166 - 177
• Main Authors: Edwards, A. Nicole, Fowlkes, Jason D., Owens, Elizabeth T., Standaert, Robert F., Pelletier, Dale A., Hurst, Gregory B., Doktycz, Mitchel J., Morrell-Falvey, Jennifer L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-12-2009
• Subjects: AFFINITY; alpha Karyopherins - analysis; Amino Acid Sequence; BACILLUS SUBTILIS; Bacterial Proteins - analysis; BASIC BIOLOGICAL SCIENCES; Binding affinity; Cell Cycle Proteins - analysis; CELL DIVISION; CLONING; DIFFUSION; DivIVA; E. coli; Green Fluorescent Proteins - analysis; IMPORTS; IN VIVO; Live cell imaging; Molecular Sequence Data; Nuclear Localization Signals - analysis; Plasmids - genetics; Protein interaction; Protein Interaction Mapping - methods; PROTEINS; Recombinant Fusion Proteins - analysis; RECOMBINATION; RHODOPSEUDOMONAS; Rhodopseudomonas - metabolism; Rhodopseudomonas palustris; SENSITIVITY; SIMULATION; Two-Hybrid System Techniques; VECTORS; Rhodopseudomonas palustris; Binding affinity; DivIVA; Protein interaction; E. coli; Live cell imaging
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.08.015

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin α (Impα), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15μM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.