期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 406, 期 23, 页码 5639-5643出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-014-8039-6
关键词
Bioluminescent protein; Aequorin; Bioluminescence; Mutagenesis; Thermostable
资金
- National Institutes of Health
- Lucille P. Markey Chair in Biochemistry and Molecular Biology of the Miller School of Medicine of the University of Miami
- Gill Eminent Professorship from the University of Kentucky
- Research Challenge Trust Fund Fellowship from the University of Kentucky
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 A degrees 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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