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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 5639-5643 |
| Number of pages | 5 |
| Journal | Analytical and Bioanalytical Chemistry |
| Volume | 406 |
| Issue number | 23 |
| DOIs | |
| State | Published - Sep 1 2014 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
Cite this
Aequorin mutants with increased thermostability. / Qu, Xiaoge; Rowe, Laura; Dikici, Emre; Ensor, Mark; Daunert, Sylvia.
In: Analytical and Bioanalytical Chemistry, Vol. 406, No. 23, 01.09.2014, p. 5639-5643.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Aequorin mutants with increased thermostability
AU - Qu, Xiaoge
AU - Rowe, Laura
AU - Dikici, Emre
AU - Ensor, Mark
AU - Daunert, Sylvia
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.
UR - http://www.scopus.com/inward/record.url?scp=85027924488&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85027924488&partnerID=8YFLogxK
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 -