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Keywords = Metridia luciferase

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15 pages, 2318 KiB  
Article
Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase
by Svetlana V. Markova, Marina D. Larionova, Igor A. Korotov and Eugene S. Vysotski
Life 2023, 13(5), 1222; https://doi.org/10.3390/life13051222 - 21 May 2023
Viewed by 2384
Abstract
Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding [...] Read more.
Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins. Full article
(This article belongs to the Special Issue Recent Advances in Bioluminescence)
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16 pages, 2649 KiB  
Article
The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins
by Marina D. Larionova, Svetlana V. Markova, Nina V. Tikunova and Eugene S. Vysotski
Int. J. Mol. Sci. 2020, 21(14), 4971; https://doi.org/10.3390/ijms21144971 - 14 Jul 2020
Cited by 8 | Viewed by 3689
Abstract
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of [...] Read more.
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins. Full article
(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications)
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