Journal
BIOTECHNOLOGY JOURNAL
Volume 11, Issue 1, Pages 91-99Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201500189
Keywords
ANL enzyme superfamily; Enzyme engineering; Firefly luciferase; Protein-protein interaction
Funding
- SENTAN, JST, Japan
- JSPS, Japan [15H04191]
- Leave a Nest Microtech Nichion award
- Kikkoman Co.
- Grants-in-Aid for Scientific Research [15H04191] Funding Source: KAKEN
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Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely a Donor that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an Acceptor that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based K-m value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to similar to 40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.
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