• Source: Protein-fragment complementation assay

Within the field of molecular biology, a protein-fragment complementation assay, or PCA, is a method for the identification and quantification of protein–protein interactions. In the PCA, the proteins of interest ("bait" and "prey") are each covalently linked to fragments of a third protein (e.g. DHFR, which acts as a "reporter"). Interaction between the bait and the prey proteins brings the fragments of the reporter protein in close proximity to allow them to form a functional reporter protein whose activity can be measured. 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 to form a functional protein may be used in a PCA. The two fragments however have low affinity for each other and must be brought together by other interacting proteins fused to them (often called "bait" and "prey" since the bait protein can be used to identify a prey protein, see figure). The protein that produces a detectable readout is called "reporter". Usually enzymes which confer resistance to nutrient deprivation or antibiotics, such as dihydrofolate reductase or beta-lactamase respectively, 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 following proteins have been used in split protein PCAs:

Beta-lactamase
Dihydrofolate reductase (DHFR)
Focal adhesion kinase (FAK)
Gal4, a yeast transcription factor (as in the classical yeast two-hybrid system)
GFP (split-GFP), e.g. EGFP (enhanced green fluorescent protein)
Horseradish peroxidase
Infrared fluorescent protein IFP1.4, an engineered chromophore-binding domain (CBD) of a bacteriophytochrome from Deinococcus radiodurans
LacZ (beta-galactosidase)
Luciferase, including ReBiL (recombinase enhanced bimolecular luciferase) and Gaussia princeps luciferase. Commercial products using luciferase include NanoLuc and NanoBIT. A modification has also been developed for lipid droplet-associated interactions.
TEV (Tobacco etch virus protease)
Ubiquitin




Genome-wide applications


The methods mentioned above have been applied to whole genomes, e.g. yeast or syphilis bacteria.




References






Further reading

Kata Kunci Pencarian:

• Protein-fragment complementation assay
• Two-hybrid screening
• Protein methods
• PCA
• Protein–protein interaction
• Bimolecular fluorescence complementation
• Protein–protein interaction prediction
• Proteome
• PFCA
• Green fluorescent protein