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. 2021 Apr 28;11:9136. doi: 10.1038/s41598-021-88315-3

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Reconstitution of split luciferase can be tuned by CC stability. Luciferase activity was determined 48 h after transfection of HEK293T cells with plasmids expressing nLuc:N8 or nluc:N6 (30 ng); and cLuc tethered to N7, P7A, P7, P7SN or to N5, P5A, P5, P5SN (10–90 ng). (A, B) Increasing amounts of plasmids expressing cLuc:CC resulted in increased luciferase activity. (C, D) Only orthogonal peptide variants with complementary electrostatic motifs formed functional luciferase. HEK293T cells were transfected with plasmids expressing nLuc:N8 or nluc:N6 (30 ng); and cLuc tethered to N7, P7A, P7, P7SN or to N5, P5A, P5, P5SN (10 ng).The values represent the means (± s.d.) from four independent cell cultures, individually transfected with the same mixture of plasmids, and are representative of two independent experiments. Amounts of used plasmids are indicated in Table S2.