Protein-Fragment Complementation Assays: Advancing Protein-Protein Interaction Analysis in Living Cells

What is the purpose of complementation assay?

Complementation assays serve the purpose of detecting protein-protein interactions that transpire within the vivacious confines of living cells. These interactions are pivotal in manifold biological processes, ranging from signal transduction to enzyme regulation and gene expression. Exploring the ins and outs of protein-protein interactions permits researchers to glean an improved comprehension of the fundamental mechanisms governing these processes, and subsequently pinpoint potential drug development targets.

What is protein-fragment complementation assay?

The protein-fragment complementation assay (PCA) is a cutting-edge technique that operates by reassembling a functional protein from multiple non-functional protein fragments. This innovative approach involves the fusion of target proteins with complementary fragments of a reporter protein - such as luciferase or green fluorescent protein (GFP) - to illuminate and highlight the interplay between them. Upon the interaction of target proteins, the fragments of the reporter protein unite nearby, consequently reconstructing the functional protein, and ultimately producing a detectable signal that can be analyzed and measured.

Protein-fragment complementation assay (PCA)Protein-fragment complementation assay (PCA) (Jaeger et al., 2014)

How does protein fragment complementation assay work?

There are several types of protein-fragment complementation assays, each with its advantages and limitations. One of the most commonly used is the bioluminescent protein-fragment complementation assay (BPCA), which relies on the reconstitution of firefly luciferase. If the target proteins interact, the fragments of luciferase are brought into proximity, leading to the reconstitution of the functional enzyme and the emission of light.

Another type of protein-fragment complementation assay is the fluorescent protein fragment-complementation assay (FPCA), which relies on the reconstitution of green fluorescent protein (GFP).

In vitro protein fragment complementation assays (IVPCA) are another type of PCA that can be used to study protein-protein interactions outside of living cells. In this essay, the target proteins are expressed and purified, and the complementary fragments of the reporter protein are added to the reaction mixture. If the target proteins interact, the fragments of the reporter protein are brought into proximity, leading to the reconstitution of the functional protein and the generation of a detectable signal.

Luciferase protein fragment complementation assay (LPCA) is a type of PCA that relies on the reconstitution of luciferase. This assay is similar to BPCA, but it uses a different type of luciferase, such as Renilla luciferase. LPCA can be used to study protein-protein interactions in vivo or in vitro, and it can be adapted to high-throughput screening applications.

Application of protein-fragment complementation assays in cell biology

PCAs are endowed with a plethora of applications in the field of cell biology, whereby the complexity of the underlying mechanisms requires sophisticated and multifaceted approaches. A key area of study involves signal transduction pathways, which are intricately intertwined with numerous biological processes, encompassing cell growth, differentiation, and apoptosis, to name a few. Through the utilization of PCA, researchers can delve into the interactions between proteins participating in these pathways, thereby unraveling their underlying mechanisms and identifying potential drug targets.

Principle of the protein complementation assay (PCA) and its applications in large-scale interaction screensPrinciple of the protein complementation assay (PCA) and its applications in large-scale interaction screens (Jia et al., 2023).

Enzyme regulation constitutes another crucial aspect of the study in which protein-fragment complementation assays have been utilized. Innumerable enzymes are modulated by their interactions with other proteins, and the aberration of these interactions can culminate in grave pathologies, including cancer and metabolic disorders. Employing PCA, researchers can pinpoint the proteins that partake in the interactions with enzymes, gaining crucial insights into the mechanisms of enzyme regulation and paving the way for the development of novel therapies targeting these debilitating diseases.

Moreover, protein-fragment complementation assays have also been leveraged to screen small molecules, evaluating their ability to interfere with protein-protein interactions in cell biology. This pioneering approach has yielded a plethora of groundbreaking discoveries, facilitating the identification of small molecules that can disrupt protein-protein interactions implicated in multifarious pathologies, including but not limited to cancer and Alzheimer's disease. Thus, this approach has undoubtedly provided a rich platform for the development of innovative therapeutic strategies.


  1. Jaeger, Werner C., et al. "Biophysical detection of diversity and bias in GPCR function." Frontiers in Endocrinology 5 (2014): 26.
  2. Jia, Yunlong, et al. "A live cell protein complementation assay for ORFeome-wide probing of human HOX interactomes." Cells 12.1 (2023): 200.
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