Part:BBa_K3168009
NanoLuc-mNeonGreen
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 normally a blue luminescent output occurs. This part consists of NanoLuc which is fused to mNeonGreen, a tetrameric fluorescent protein derived from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is three to five times brighter than GFP and is more stable and less vulnerable to laser induced bleaching (Shaner, 2013). Because the emission spectrum of NanoLuc and the excitation spectrum of mNeonGreen overlap, bioluminescence resonance energy transfer (BRET) occurs and the green emission from mNeonGreen is observed instead, without further external illumination (figure 1). The closer the donor and acceptor are, the more efficient the BRET is. Therefore, the C terminus of mNeonGreen overlaps with the N terminus of NanoLuc, no linker is used. A His-tag in the front and a Strep-tag at the end are included for protein purification.
Figure 1. Schematic representation BRET between the donor NanoLuc and the acceptor mNeonGreen.
Usage and Biology
This fusion protein allows ratiometric measurements where NanoLuc-mNeonGreen acts as a calibrator luciferase to allow for time and concentration independent measurements.
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.
Shaner, N.C., et al. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods. 10(5), 407-409.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 245
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 739
- 1000COMPATIBLE WITH RFC[1000]
| None |