title: Expression of a soluble truncated Vargula luciferase in Escherichia coli.
abstract: Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA; University of Miami, Department of Chemistry, Coral Gables, 33146, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Hunt
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA; University of Miami, Department of Chemistry, Coral Gables, 33146, USA.
authors.forename: Angeliki
authors.initials: A
authors.lastname: Moutsiopoulou
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA.
authors.forename: David
authors.initials: D
authors.lastname: Broyles
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA.
authors.forename: Trajen
authors.initials: T
authors.lastname: Head
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA.
authors.forename: Emre
authors.initials: E
authors.lastname: Dikici
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
authors.affiliation: University of Miami, Leonard M. Miller School of Medicine, Department of Biochemistry & Molecular Biology, Miami, 33136, USA. Electronic address: sdeo@med.miami.edu.
authors.forename: Sapna K
authors.initials: SK
authors.lastname: Deo
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003445:Crustacea / Q000201:enzymology / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D060829:Arthropod Proteins; D011994:Recombinant Proteins; D008156:Luciferases
keywords: Bacterial expression; Bioluminescence; Truncated protein; Vargula luciferase