Creative Biolabs supplies highly sensitive Protein-fragment Complementation Assay (PCA) service to detect dynamic protein-protein interactions (PPIs), conformation changes, and protein complex dimensions in virtually any cell type or organism.
Protein-Fragment Complementation Assay (PCA)
In PCA, protein A and B, two proteins of interest, is fused to one complementary fragment of a rationally dissected reporter protein, respectively. If the protein A and B could interact, the reporter fragments are taken together, fold and reconstitute its native structural activity. The reporter proteins have been picked as those generating a detectable signal, including fluorescent, luminescent, colorimetric signal, and survival selection assay. Owing to its high sensitivity, high signal-to-background ratio, as well as full reversibility, PCA is an extremely powerful and widely used approach to monitor direct and proximal associations between proteins expressed at endogenous level in any intact living cell, in subcellular compartments, in vivo or in vitro. The reversibility of PCA assures accurate measurements of the kinetics and equilibrium aspects of PPI assembly or disassembly.
In addition to the above mentioned, Creative Biolabs also displays the following capabilities of PCA: the detection of induced versus constitutive PPIs under any conditions (including occurrence in hormone, nutritional, developmental or environmental-induced signals) and screening of cDNA libraries for PPIs in any cell type.
Fig.1 The protein-fragment complementation assay (PCA) strategy.
(spheres: interacting protein A and B; ribbons: unfolded peptide fragments)
PCA acts as a general experimental method that enables the quantitative detection of dynamic PPIs and provides structural and topological details of how a PPI is formed or if complexes go through conformation changes. From above chart, a crucial point of this approach is that the fragments are designed so that they can’t fold spontaneously; otherwise a situation would occur that results in a false-positive signal.
The Principle of PCA Strategy
In the process of PCA, PPIs are measured depending on fusing each of the proteins of interest to complementary N- or C-terminal peptides of a reporter protein that has been rationally dissected. The split reporter proteins are brought into proximity after the interaction of two interest proteins, allowing them to fold into the three-dimensional structure of the reporter protein, thus reconstituting the activity of the reporter signal.
Fig.2 Principle of Fluc PCA.
The Category of PCA
This general principle of PCA has been applied to generate assays using reporter proteins, whose activities are measured in a number of different ways, depending on the desired applications.
Typical PCA has been using dihydrofolate reductase (DHFR), β-lactamase, GFP or luciferase producing a detectable signal in split protein PCAs. When fluorescent proteins are reconstituted the PCA is called bimolecular fluorescence complementation assay (BiFC).
The DHFR-PCA can be used in numerous applications to display both simple survival-selection as readout and a fluorescent assay simultaneously allowing quantitative detection and cellular localization of protein interactions to be performed. Unlike DHFR, the β-lactamase-based PCA assay is worked as a much sensitive in vivo or in vitro quantitative detector of PPIs as one way measures the continuous conversion of substrate to fluorescent or colored product.
Fig.3 DHFR-PCA Model for the determination of PPIs in yeast cytosol.
The Features and Advantages of PCA
Creative Biolabs has completed a growing number of study services using PCA, which demonstrates the broad potential of this approach. The PCA technique not only can analyze the localization of protein molecular interaction dynamically, map intracellular signal transduction pathway and protein biochemical network but also is applied to protein library, target discovery and high throughput drug screening.