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.
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Acknowledgments
This work was supported in part by grants from the National Institutes of Health. S.D. 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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Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli.
This work was completed at University of Kentucky, Department of Chemistry, Lexington, KY 40506, USA
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Qu, X., Rowe, L., Dikici, E. et al. Aequorin mutants with increased thermostability. Anal Bioanal Chem 406, 5639–5643 (2014). https://doi.org/10.1007/s00216-014-8039-6
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DOI: https://doi.org/10.1007/s00216-014-8039-6