title: Aequorin mutants with increased thermostability.
abstract: Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY, 40506, USA.
authors.forename: Xiaoge
authors.initials: X
authors.lastname: Qu
authors.forename: Laura
authors.initials: L
authors.lastname: Rowe
authors.forename: Emre
authors.initials: E
authors.lastname: Dikici
authors.forename: Mark
authors.initials: M
authors.lastname: Ensor
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
mesh_terms: D000331:Aequorin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D006358:Hot Temperature; D007700:Kinetics; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D055550:Protein Stability
chemical_list: D000331:Aequorin
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