Authors: Xiaoge Qu Laura Rowe Emre Dikici Mark Ensor Sylvia Daunert
Publish Date: 2014/08/02
Volume: 406, Issue: 23, Pages: 5639-5643
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 nontoxicity of bioluminescent reporter systems Significant thermal stability of bioluminescent labels is essential however due to the longitudinal nature and physiological temperature conditions of many bioluminescentbased 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 275 times more of their initial bioluminescence activity than wildtype 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 farThis work was supported in part by grants from the National Institutes of Health SD is grateful for support from the Lucille P Markey Chair in Biochemistry and Molecular Biology of the Miller School of Medicine of the University of Miami as well as from a Gill Eminent Professorship from the University of Kentucky Xiaoge Qu acknowledges support from a Research Challenge Trust Fund Fellowship from the University of Kentucky
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