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 increased stability, 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 no evidence of posttranslational modifications or subcellular partitioning in mammalian cells (Hall, 2012). Furimazine, NanoLuc’s substrate, shows increased stability and lower background activity which 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, which was mutated to a serine to create 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 (Bioluminescence Resonance Energy Transfer) based sensors.

Usage and Biology

NanoLuc has potential to play a great 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]