The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells

Markova, Svetlana V.; Larionova, Marina D.; Gorbunova, Darya A.; Vysotski, Eugene S.

doi:10.1016/j.jphotobiol.2017.08.024

Cited by 7 publications

(5 citation statements)

References 28 publications

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“…The E. coli cells BL21 CodonPlus (DE3)-RIPL (Stratagene, San Diego, CA, USA), harboring constructs with MLuc7 deletion mutants, were cultivated at 37 °C in LB medium supplemented with 200 µg/mL ampicillin. The luciferase synthesis was induced at OD 600 = 1.2 with 1 mM IPTG, after induction, the cultivation was continued for 1 h. Cell harvesting, washing of inclusion body pellets, solubilization of luciferases, oxidative refolding, and chromatography purification were performed as described for MLuc7 [ 17 ] with certain modifications [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase

Markova,

Larionova,

Korotov

et al. 2023

Life

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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.

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Section: Methodsmentioning

confidence: 99%

Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase

Markova,

Larionova,

Korotov

et al. 2023

Life

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“…The more simple and cost-effective way to obtain active natively folded CopLucs was shown for MLuc7 M. longa luciferase which was recovered from insoluble aggregates synthesized in E. coli by optimizing the refolding conditions (37). Bioluminescent and structural features of this refolded MLuc7 studied in a set of identical experiments were the same as those of MLuc7 from insect cells.…”

Section: Expression and Obtaining Of Correctly Folded Copepod Lucifermentioning

confidence: 99%

“…Although CopLucs revealed the promising potential as bioluminescent labels, their application in various binding assays is still limited which is primarily caused by difficulties of obtaining the active copepod luciferases of high purity. However, we believe that the recently developed procedures of obtaining the correctly folded high‐active and high‐pure copepod luciferases from both culture medium of insect cells and inclusion bodies of E. coli cells intensify employing these luciferases as labels for various in vitro assays.…”

Section: Application Of Copepod Luciferasesmentioning

confidence: 99%

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

Photochem & Photobiology

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Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

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“…In contrast, the baculovirus expression vector system (BEVS) has been applied in the reproducible and effective production of various prokaryotic and eukaryotic proteins [ 26 , 27 , 28 ]. Since BEVS is based on eukaryotic protein expression machinery and secretes recombinant proteins in a soluble form, proteins necessary for maintaining natural protein conformation have been produced with high purity and functionality [ 29 , 30 , 31 ]. To date, several bacterial and eukaryotic toxin molecules have been successfully purified using BEVS [ 31 , 32 , 33 , 34 ]; however, to the best of our knowledge, no studies have reported the use of BEVS to produce recombinant wild-type SEB.…”

Section: Introductionmentioning

confidence: 99%

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression

Jang

Kim

et al. 2022

Biosensors

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Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

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The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells

Cited by 7 publications

References 28 publications

Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase

Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression

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