Protein-fragment complementation assay

Protein-fragment complementation assay, or PCA, is a method for the identification of protein–protein interactions in biological systems. It is used in the field of proteomics. In the protein-fragment complementation assay, the proteins of interest ("Bait" and "Prey") are each covalently linked to incomplete fragments of a third protein and are expressed in vivo (sometimes called a "reporter"). Interaction between the "bait" and the "prey" proteins brings the fragments of the "reporter" protein in close enough proximity to allow them to reform and become the functional reporter protein. This principle can be applied to many different "reporter" proteins and is also the basis for the yeast two-hybrid system, an archetypical PCA assay.

Split protein assays


Any protein that can be split into two parts and reconstituted non-covalently may be used in a PCA. The two parts just have to be brought together by other interacting proteins fused to them (typically called "bait" and "prey" (see figure). The protein that produces a detectable readout is called "reporter". Usually enzymes which confer resistance to antibiotics, such as Dihydrofolate reductase or Beta-lactamase, or proteins that give colorimetric or fluorescent signals are used as reporters. When fluorescent proteins are reconstituted the PCA is called Bimolecular fluorescence complementation assay. The most popular PCAs utilize versions of the following proteins:


 * Dihydrofolate reductase
 * Beta-lactamase
 * Yeast Gal4 (as in the classical yeast two-hybrid system)
 * Split-TEV
 * Split Luciferase
 * Split-Ubiquitin
 * Split-EGFP (enhanced green fluorescent protein)