Rapid titration of recombinant baculoviruses based on NanoLuc secretion in early infection

Li, Jige; Xu, Meng; Sun, Juan; Li, Junjun; Chen, Qin; Chen, Jian

doi:10.1016/j.jviromet.2022.114565

Cited by 2 publications

(3 citation statements)

References 32 publications

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“…[ 5,6 ] However, FLuc and RLuc have large molecular weights of 62 and 36 kDa, respectively, which may impair the functions of target proteins when used as reporter proteins for the above various purposes and in certain applications, including bioluminescence resonance energy transfer (BRET) and protein complementary analysis (PCA, or split Luc assay), [ 7 ] and may also affect their expression in genomic insertions such as in recombinant reporter viruses. [ 8 ] Therefore, recent interests in bioluminescence applications have focused on smaller and brighter luciferases.…”

Section: Introductionmentioning

confidence: 99%

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

Ohmuro‐Matsuyama

Matsui

Kanai

et al. 2023

Biotechnology Journal

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Luciferases are widely used as reporter proteins in diverse fields from basic biology to medical and environmental researches. Development of luciferase applications for reporter proteins requires small size without target inhibition, appropriate genomic insertion for high expression level, and bright emission for detection sensitivity. We previously developed the minimal luciferase picALuc, but its luminescence was still dim compared to other bright luciferases in terms of expression in Escherichia coli. In this study, diverse additions of oligopeptides with charged residues (eight amino acids in total) to the C-terminus of picALuc enhanced luminescence by up to approximately 50fold, that is, enhanced enzymatic activity. Moreover, these high luminescence activities were achieved in bacterial and mammalian expression, suggesting their further applicability in many expression systems. The finding in this study that the simple addition of oligopeptides with charged residues (or charge engineering of this kind) enhances enzymatic activity may be applied to a wide variety of enzymatic reactions and protein functions.

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Section: Introductionmentioning

confidence: 99%

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

Ohmuro‐Matsuyama

Matsui

Kanai

et al. 2023

Biotechnology Journal

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“…The brightness, thermostability, and small size of reporter proteins make them useful in applications. Their small size can reduce steric hindrance and misfolding of reporter-fused proteins [3,4], making them amenable to genome insertion [5] and drug screening [6]. Additionally, the use of small luciferases can yield higher signals in bioluminescence resonance energy transfer (BRET)-based assays [7].…”

Section: Introductionmentioning

confidence: 99%

“…led to a luciferase without disulfide bonds (from nuclear transport factor 2-like superfamily), making it useful in intracellular expression systems. Conversely, picALuc is suited to secretary expression systems for analyzing recombinant viruses [5] and detecting disease markers via application of secretary reporters in blood and urine [14][15][16]. Finally, the latest version of picA-Luc (picALuc2.0) features a remarkably improved luminescence intensity (approximately 25-50-fold), achieved by adding a charged oligopeptide [17].…”

Section: Introductionmentioning

confidence: 99%

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Ohmuro‐Matsuyama,

Matsui,

Kanai

et al. 2023

The FEBS Journal

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Luciferases are widely used as reporter proteins in various fields. Recently, we developed a minimal bright luciferase, picALuc, via partial deletion of the artificial luciferase (ALuc) derived from copepods luciferases. However, the structures of copepod luciferases in the substrate‐bound state remain unknown. Moreover, as suggested by structural modeling, picALuc has a larger active site cavity, unlike that in other copepod luciferases. Here, to explore the bioluminescence mechanism of picALuc and its luminescence properties, we conducted multiple mutational analyses, and identified residues and regions important for catalysis and bioluminescence. Mutations of residues likely involved in catalysis (S33, H34, and D55) markedly reduced bioluminescence, whereas that of residue (E50) (near the substrate in the structural model) enhanced luminescence intensity. Furthermore, deletion mutants (Δ70–Δ78) in the loop region (around I73) exhibited longer luminescence lifetimes (~30 min), and were reactivated multiple times upon re‐addition of the substrate. Due to the high thermostability of picALuc, one of its representative mutant (Δ74), was able to be reused, i.e., luminescence recycling, for day‐scale time at room temperature. These findings provide important insights regarding picALuc bioluminescence mechanism and copepod luciferases, and may help with sustained observations in a variety of applications.

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Rapid titration of recombinant baculoviruses based on NanoLuc secretion in early infection

Cited by 2 publications

References 32 publications

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

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