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The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter

Paper ID Volume ID Publish Year Pages File Format Full-Text
23630 43459 2012 8 PDF Available
Title
The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter
Abstract

Bacterial luciferase from Vibrio campbellii is a thermostable enzyme with an in vitro thermal inactivation half-life of ∼1020 min at 37 °C. The enzyme also binds tightly to reduced FMN. In this study, a V. campbellii fusion luciferase construct in which the α and β subunits are linked with a decapeptide was made and characterized. In general, the overall enzymatic properties of the two enzymes are similar. Expression of the enzymes in Escherichia coli demonstrated that the V. campbellii fusion luciferase emits less light than the native luciferase, but still emits a much greater amount of light than native luciferase from Vibrio harveyi and Photobacterium leiognathi TH1. The intensity of light emitted by the V. campbellii fusion luciferase was more than 80-fold greater than that from the V. harveyi native luciferase when expressed at 37 °C. Biochemical characterization has shown that the V. campbellii fusion luciferase also retains a high binding affinity for reduced flavin mononucleotide and high thermostability. The levels of bioluminescence emitted by the V. campbellii fusion luciferase expressed in HEK293T cells reached ∼1 × 106 Relative Light Units/mg total protein. These findings suggest that the V. campbellii fusion luciferase is a promising candidate for further development as a luciferase-based reporter for eukaryotic systems.

► The fusion form of Vibrio campbellii luciferase can efficiently catalyze the reaction. ► The fusion luciferase is stable at 37 °C and binds tightly to flavin. ► The fusion enzyme is suitable for bioreporter applications. ► The fusion luciferase can be expressed in HEK293T cells.

Keywords
C1, NADH-dependent reductase component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii; LuxAB, bacterial luciferase; FMN, flavin mononucleotide; FMNH−, reduced form of FMN; HPA, p-hydroxyphenylacetate; Kd, dissociation constant; kobs, a
First Page Preview
The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 162, Issues 2–3, 31 December 2012, Pages 346–353
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering