TY - JOUR
T1 - Aequorin mutants with increased thermostability
AU - Qu, Xiaoge
AU - Rowe, Laura
AU - Dikici, Emre
AU - Ensor, Mark
AU - Daunert, Sylvia
N1 - Funding Information:
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.
PY - 2014/9/1
Y1 - 2014/9/1
N2 - 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.
AB - 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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U2 - 10.1007/s00216-014-8039-6
DO - 10.1007/s00216-014-8039-6
M3 - Article
C2 - 25084737
AN - SCOPUS:85027924488
VL - 406
SP - 5639
EP - 5643
JO - Fresenius Zeitschrift fur Analytische Chemie
JF - Fresenius Zeitschrift fur Analytische Chemie
SN - 0016-1152
IS - 23
ER -