{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T04:54:43Z","timestamp":1773377683938,"version":"3.50.1"},"reference-count":38,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2023,7,13]],"date-time":"2023-07-13T00:00:00Z","timestamp":1689206400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Society for the Promotion of Science (JSPS)","award":["21H04948"],"award-info":[{"award-number":["21H04948"]}]},{"name":"Japan Society for the Promotion of Science (JSPS)","award":["20K21851"],"award-info":[{"award-number":["20K21851"]}]},{"name":"Japan Society for the Promotion of Science (JSPS)","award":["17H01215"],"award-info":[{"award-number":["17H01215"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors\u2019 previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.<\/jats:p>","DOI":"10.3390\/s23146376","type":"journal-article","created":{"date-parts":[[2023,7,14]],"date-time":"2023-07-14T00:49:30Z","timestamp":1689295770000},"page":"6376","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Creation of Artificial Luciferase 60s from Sequential Insights and Their Applications to Bioassays"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7033-9056","authenticated-orcid":false,"given":"Sung-Bae","family":"Kim","sequence":"first","affiliation":[{"name":"Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8569, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4568-9043","authenticated-orcid":false,"given":"Tadaomi","family":"Furuta","sequence":"additional","affiliation":[{"name":"School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3104-6963","authenticated-orcid":false,"given":"Nobuo","family":"Kitada","sequence":"additional","affiliation":[{"name":"Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu 182-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9741-0166","authenticated-orcid":false,"given":"Shojiro A.","family":"Maki","sequence":"additional","affiliation":[{"name":"Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu 182-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,13]]},"reference":[{"key":"ref_1","unstructured":"Shimomura, O. (2006). Bioluminescence, World Scientific Publishing Co., Pte. Ltd."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4438","DOI":"10.1073\/pnas.88.10.4438","article-title":"Isolation and expression of a cDNA encoding Renilla reniformis luciferase","volume":"88","author":"Lorenz","year":"1991","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4378","DOI":"10.1021\/ac025742k","article-title":"Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization","volume":"74","author":"Verhaegent","year":"2002","journal-title":"Anal. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3212","DOI":"10.1074\/jbc.M309639200","article-title":"Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme","volume":"279","author":"Markova","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1271\/bbb.68.565","article-title":"CDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca","volume":"68","author":"Nakajima","year":"2004","journal-title":"Biosci. Biotech. Bioch."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1016\/0003-9861(79)90288-1","article-title":"Dinoflagellate Luciferases-Purification of Luciferases from Gonyaulax-Polyedra, Pyrocystis-Lunula, and Pyrocystis-Fusiformis","volume":"196","author":"Lecuyer","year":"1979","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/S0014-5793(00)01963-3","article-title":"Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase","volume":"481","author":"Inouye","year":"2000","journal-title":"FEBS Lett."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"233","DOI":"10.2116\/analsci.20R003","article-title":"Bioluminescent Imaging Systems for Assay Developments","volume":"37","author":"Kim","year":"2021","journal-title":"Anal. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"14955","DOI":"10.1073\/pnas.2432563100","article-title":"Darwinian natural selection for orange bioluminescent color in a Jamaican click beetle","volume":"100","author":"Stolz","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","first-page":"1098","article-title":"Bioluminescence imaging: Basics and practical limitations","volume":"1098","author":"Badr","year":"2014","journal-title":"Methods Mol. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1038\/nmeth1070","article-title":"Red-shifted Renilla reniformis luciferase variants for imaging in living subjects","volume":"4","author":"Loening","year":"2007","journal-title":"Nat. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1126\/science.aaq1067","article-title":"Single-cell bioluminescence imaging of deep tissue in freely moving animals","volume":"359","author":"Iwano","year":"2018","journal-title":"Science"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"8732","DOI":"10.1021\/ac2021882","article-title":"Superluminescent variants of marine luciferases","volume":"83","author":"Kim","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1093\/protein\/gzs016","article-title":"Labor-effective manipulation of marine and beetle luciferases for bioassays","volume":"25","author":"Kim","year":"2012","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1021\/bc400411h","article-title":"Creation of artificial luciferases for bioassays","volume":"24","author":"Kim","year":"2013","journal-title":"Bioconjugate Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"594","DOI":"10.1021\/acscombsci.7b00081","article-title":"Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools","volume":"19","author":"Kim","year":"2017","journal-title":"ACS Comb. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.bbrc.2007.10.152","article-title":"Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps","volume":"365","author":"Inouye","year":"2008","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.gene.2008.07.041","article-title":"Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica","volume":"425","author":"Takenaka","year":"2008","journal-title":"Gene"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1415","DOI":"10.2116\/analsci.25.1415","article-title":"Determination of the Androgenicity of Ligands Using a Single-chain Probe Carrying Androgen Receptor N-Terminal Peptides","volume":"25","author":"Kim","year":"2009","journal-title":"Anal. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1835","DOI":"10.1021\/bc200220k","article-title":"A Bioluminescent Probe for Salivary Cortisol","volume":"22","author":"Kim","year":"2011","journal-title":"Bioconjugate Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1039\/C9CC07634D","article-title":"Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice","volume":"56","author":"Kim","year":"2020","journal-title":"Chem. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"30205","DOI":"10.1074\/jbc.270.50.30205","article-title":"Phosphorylation of tyrosine 537 on the human estrogen receptor is required for binding to an estrogen response element","volume":"270","author":"Arnold","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/S0960-0760(02)00264-9","article-title":"Sex steroid hormones act as growth factors","volume":"83","author":"Migliaccio","year":"2002","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2345","DOI":"10.1021\/acschembio.7b00454","article-title":"Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications","volume":"12","author":"Gaur","year":"2017","journal-title":"ACS Chem. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1016\/j.ymthe.2004.10.016","article-title":"Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo","volume":"11","author":"Tannous","year":"2005","journal-title":"Mol. Ther."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2219","DOI":"10.1038\/s41598-021-81430-1","article-title":"Color-tunable bioluminescence imaging portfolio for cell imaging","volume":"11","author":"Tamaki","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"20069","DOI":"10.1038\/s41598-020-76486-4","article-title":"Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR","volume":"10","author":"Wu","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1021\/acschembio.5b00753","article-title":"NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells","volume":"11","author":"Dixon","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1021\/cb800004s","article-title":"An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule","volume":"3","author":"Kim","year":"2008","journal-title":"ACS Chem. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1016\/j.bbrc.2014.04.128","article-title":"Functional artificial luciferases as an optical readout for bioassays","volume":"448","author":"Kim","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1042\/bj3060537","article-title":"Cause of Spectral Variation in the Luminescence of Semisynthetic Aequorins","volume":"306","author":"Shimomura","year":"1995","journal-title":"Biochem. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1039\/C4CC06886F","article-title":"Bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution","volume":"51","author":"Nishihara","year":"2015","journal-title":"Chem. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1039\/c5pp00456j","article-title":"Lighting up bioluminescence with coelenterazine: Strategies and applications","volume":"15","author":"Jiang","year":"2016","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1848","DOI":"10.1021\/cb3002478","article-title":"Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate","volume":"7","author":"Hall","year":"2012","journal-title":"ACS Chem. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2324","DOI":"10.1021\/bc900330w","article-title":"Molecular Tension-Indexed Bioluminescent Probe for Determining Protein-Protein Interactions","volume":"20","author":"Kim","year":"2009","journal-title":"Bioconjugate Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1021\/acs.bioconjchem.5b00421","article-title":"Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells","volume":"27","author":"Kim","year":"2016","journal-title":"Bioconjugate Chem."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/14\/6376\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:12:19Z","timestamp":1760127139000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/14\/6376"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,7,13]]},"references-count":38,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["s23146376"],"URL":"https:\/\/doi.org\/10.3390\/s23146376","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,7,13]]}}}