The pNL3.2.CMV Vector constitutively expresses the NanoLuc® reporter fused to a PEST destabilization domain (NlucP). The NlucP fusion protein naturally accumulates at low intracellular levels due to constitutive proteosomal degradation, thus serving as a negative control for experiments configured to measure regulated changes in NanoLuc® luciferase expression levels (e.g., NanoLuc® fusion vectors). This vector also encodes a hygromycin resistance gene for selection in mammalian cells.
NanoLuc® (Nluc) luciferase is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. The luminescent reaction is ATP-independent and designed to suppress background luminescence for maximal assay sensitivity.
About the NanoLuc® Luciferase Reporter Enzyme
NanoLuc® luciferase in the form of NanoLuc®-PEST (NlucP) closely couples protein expression to changes in transcriptional activity and increased signal-to background ratios. The PEST sequence appended to Nluc destabilizes the reporter protein, make NanoLuc® luciferase more responsive to changes in the cell. Luminescence is linearly proportional to the amount of NanoLuc® protein over a 1,000,000-fold concentration range, with a signal half-life ≥2 hours when detected with Nano-Glo® Luciferase Assay Reagent.
NanoLuc® luciferase possesses a number of physical properties that make it an excellent reporter protein:
- very small, monomeric enzyme (171 amino acids; 513bp)
- high thermal stability (Tm = 60°C)
- active over a broad pH range (pH 6–8)
- no post-translational modifications or disulfide bonds
- uniform distribution in cells
- emission spectrum well suited for bioluminescence resonance energy transfer (BRET; λmax = 465nM).
The pNL vector series uses a pGL4-based backbone for easy sequence transfer from existing plasmids. This backbone design also reduces anomalous results by removing many transcription factor binding sites and other potential regulatory elements. The NlucP gene is codon optimized and has had many potential regulatory elements or other undesirable features removed (such as common restriction enzyme sites).
pNL3.2.CMV Vector GenBank® Accession Number KF853603 and vector sequence text file.