Part:BBa_K3168006
Cysteine-free-NanoLuc
NanoLuc is a deep-sea shrimp derived luciferase, which is smaller compared to the Firefly and Renilla luciferases and therefore offers certain advantages over the traditional methods. NanoLuc has an increased stability, a smaller size and a >150-fold increase in luminescence (England, 2016). Furthermore, NanoLuc displays high physical stability, maintains its activity during incubation up to 55 oC or in culture medium for >15 h at 37 oC and shows in mammalian cells no evidence of posttranslational modifications or subcellular partitioning (Hall, 2012). The substrate for NanoLuc, called Furimazine shows increased stability and lower background activity, this enhances the possibilities for bioluminescence imaging (England, 2016). Furimazine reacts with NanoLuc in the presence of oxygen. Furimazine is converted to Furimamide and a blue luminescence output occurs. Furthermore, a flexible (SGG)2 linker is located in front of NanoLuc to enable the formation of fusion proteins. A strep tag is also included at the end for protein purification.
NanoLuc originally contains one cysteine, this cysteine was mutated to a serine, which results in a cysteine free construct. Hereby, this NanoLuc can be used in the formation of fusion proteins which can be labeled via maleimide coupling. This is useful for BRET (Bioluminescent Resonance Energy Transfer) based sensors.
Usage and Biology
NanoLuc has a huge potential role in disease detection, molecular imaging, and therapeutic monitoring (England, 2016). NanoLuc is used a lot in the development of BRET-sensors (Arts, 2017).
References
Arts, R., Aper, S. J., & Merkx, M. (2017). Engineering BRET-sensor proteins. In Methods in enzymology (Vol. 589, pp. 87-114). Academic Press.
England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjugate chemistry, 27(5), 1175-1187.
Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., ... & Robers, M. B. (2012). Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS chemical biology, 7(11), 1848-1857.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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