Splitkomplementaarisuus

Split complementation refers to a set of molecular biology techniques in which a functional protein is divided into two nonfunctional fragments that can reassemble to restore activity when brought into proximity. The approach is widely used to study protein–protein interactions, signaling events, and cellular localization. Common implementations include fluorescent reporters such as split GFP or split YFP, and enzymatic reporters such as split luciferase or split beta-galactosidase. These systems enable detection of interactions in living cells or organisms and can be adapted for high-throughput screening.

In a typical experiment, the N-terminal fragment is fused to one protein of interest (protein A) and the C-terminal fragment to another (protein B). If A and B interact, the fragments are co-localized and reconstitute a detectable signal—fluorescence for reporter proteins or luminescence for enzyme-based reporters. Important design considerations include the choice of split site, linker length and composition, fragment stability, and the potential for spontaneous assembly. Some split systems are designed to be reversible, while others, such as many BiFC (bimolecular fluorescence complementation) variants, form stable, often irreversible complexes, yielding persistent signals even after the interaction ends.

Advantages of split complementation include direct visualization of molecular interactions in living cells and compatibility with standard microscopy or plate-based assays. Limitations involve potential background signal from non-specific assembly, overexpression artifacts, and the irreversibility of some systems, which can complicate kinetic analyses. Proper controls, such as non-interacting partners or non-functional fragment mutants, are essential. Split complementation is widely used to map interaction networks, monitor signaling dynamics, and facilitate drug discovery through interaction-based readouts.