Novel PPI assay using functional complementation of mutant firefly luciferases

Firefly luciferase catalyzes series of reactions to efficiently yield its bioluminescence using two substrates luciferin and ATP. This reaction process can be largely divided into two half reactions: adenylation of luciferin and oxidative reactions using luciferyl adenylate (LH2-AMP).

The luciferase, as a member of acyl-adenylate enzyme superfamily, is composed of two (N-terminal and C-terminal) domains as well as other enzymes, and the two domains has been thought to cooperate together for the light-emitting reactions.

We found that although weak (and slow), the N-terminal domain alone has its luminescence activity using the two substrates, and also found that we can specifically and sensitively detect the reaction intermediate LH2-AMP in the presence of excess luciferin and ATP by using this domain based on the different kinetics. This enabled us to measure the LH2-AMP concentration during the reactions of various mutants, which is expected to lead to the elucidation of the not fully understood reaction mechanism. Also expected is the development of protein-protein interaction detection method based on the mutant enzymes (FlimPIA).

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