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. 2020 Jun 29;10:10522. doi: 10.1038/s41598-020-67468-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Reaction-induced inactivation of Gaussia luciferase enables the use of a single construct as both a bait and a prey. (a) Pentameric prey proteins can be used as a bait for sensitive interaction detection. Biotinylated Cd200R Gaussia luciferase-tagged pentamer (filled circles) or monomers (empty circles) were serially diluted and immobilised on a streptavidin-coated microtitre plate as bait proteins and probed for interactions with a beta-lactamase-tagged Cd200 pentamer. (b) Coelenterazine was added to a dilution series of purified Cd200 Gaussia luciferase pentamer which elicited a strong luminescence signal that was gradually lost after around an hour. A second administration of substrate did not generate the same bright luminescence signal. (c) Gaussia enzymatic activity is not restored after a nickel-affinity purification of the inactivated luciferase. Reaction inhibited Gaussia luciferase was purified using Ni–NTA agarose beads with several wash steps before addition of fresh coelenterazine (100 pmol). (d) Pentameric (non-biotinylated) Cd200 Gaussia luciferase prey was probed against biotinylated and inactivated pentameric Cd200R Gaussia luciferase bait (filled circles). The Cd200 prey was also probed against a reaction-inhibited pentameric Gaussia luciferase Cd200 protein used as a non-interacting control bait (empty circles). The inflection point for each interpolated curve is displayed as the log baits (moles) value on each graph. Representative experiments shown with n = 3 for each quantity of prey with SEM for each data point and the 95% CI for the interpolated curves.