1. Field of the Invention
The present invention relates to a method for detecting protein-protein interactions in living cells, and more particularly, to a method for providing cells comprising a first construct and a second construct, wherein the first construct comprises a polynucleotide encoding a first fusion protein which comprises a bait protein, a first fluorescent protein and a CBD (cellulose-binding domain) protein, and wherein the second construct comprises a polynucleotide encoding a second fusion protein which comprises a prey protein and a second fluorescent protein so as to allow formation of inclusion bodies, and detecting interactions between the bait protein and the prey protein that are displayed by inclusion bodies, a method for isolating the prey protein bound to the bait protein using the cells comprising the constructs, the prepared cells, and a kit for detecting protein-protein interactions comprising the constructs.
2. Description of the Related Art
Many functions of organisms are regulated by a network of intermolecular interactions. Thus, understanding of protein-protein interactions (PPI) in cells is an important issue in current biological research. PPI has been mainly examined by in vitro experiments such as immunoprecipitation and tandem affinity purification (TAP). However, there are limitations to the in-vitro approach for the analysis of dynamic protein-protein interactions in-vivo. In addition, an yeast two-hybrid (Y2H) system has been used for highly sensitive detection of PPIs under in vivo conditions. However, a Y2H system is disadvantageous in that it must be performed in yeast, nuclear translocation is required, and it relies on the indirect readout of secondary induced transcriptional activation and effects, not in direct contact, resulting in false-positives.
In order to overcome the above disadvantages, numerous methods have been developed, based on fluorescence techniques such as FRET (fluorescence resonance energy transfer), fluorescence complementation assay, and fluorescence co-localization. Among them, FRET is a method of detecting changes in fluorescence spectrum when a donor protein and an acceptor protein are within a distance of 2-8 nm, in which direct excitation of the donor molecule results in resonance energy transfer to the acceptor molecule, provided two fluorescent molecules at different wavelength are adjacent to each other. Fluorescence complementation assay is a technique of visualizing protein-protein interactions in living cells, based on fluorescence recovery due to complementation of GFP fragments, in which a fluorescent protein is split into N- and C-terminal fragments, the fragment is attached to each binding protein and expressed in the cell, and fusion of the two proteins results in reconstitution of N- and C-terminal fragments of the fluorescent protein that could be visualized. Even though the two methods are useful for the analysis of protein interactions in animal cells using a high speed sorter flow cytometry, the methods are difficult to apply for bacterial cells. For example, the detection level of FRET is too low to detect weak signals from bacterial cells considerably smaller than eukaryotic cells. Fluorescence complementation assay may show non-uniform protein expression in cells of a large size, and is influenced by other factors in bacterial cells, leading to false-positive results.
Fluorescence co-localization is used by localization in particular organelles of eukaryotic cells, but cannot be applied to a bacterial system that has no cell organelles. A recent study reported that a fluorescent PPI complex formed by co-expression of a bait protein-fused cell division protein and a fluorescent prey protein is recruited to the cell pole of E. coli (Edwards, A. N., et al., 2009. An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities. Anal. Biochem. 395:166-77.).
The present inventors have made many efforts to develop a method for analyzing interactions between biomolecules in living cells. As a result, they found that Cellulomonas fimi-derived family II cellulose-binding domain (CBD) is self-aggregated to form inclusion bodies (IB) in E. coli, and CBD-induced IB has a feature of capturing a particular interacting protein in the particles when the interacting proteins are co-expressed. Accordingly, the present inventors intended to develop a new fluorescence co-localization method by using the inclusion bodies as artificial cell organelles in bacterial cells as well as eukaryotic cells. Finally, the present inventors found that an antiparallel leucine zipper is used as a model of interacting proteins to observe fluorescence co-localization to inclusion bodies (FCIB) using a fluorescence microscope and a high speed sorter flow cytometer so that interactions between the proteins having low binding affinity can be analyzed and isolated, thereby completing the present invention.
USPTO Backgrounds
USPTO Backgrounds