Description
The pNLF1 Vectors use traditional cloning with a multiple cloning site (MCS) to generate N- or C-terminal fusions to NanoLuc® luciferase. Create full-length Nluc protein fusions using the pNLF1-N [CMV/Hygro] Vector (N terminus) and pNLF1-C [CMV/Hygro] Vector (C terminus). In addition, attach secreted Nluc to the N terminus of the protein of interest using the pNLF1-secN [CMV/Hygro] Vector. All vectors contain a mammalian selectable marker to create a stable line.
The small size (19.1kDa) and extreme brightness of NanoLuc® luciferase (Nluc; about 100-fold brighter than either firefly [ Photinus pyralis ] or Renilla reniformis ) make it an ideal protein fusion partner. When visualizing intracellular protein dynamics, the bright NanoLuc® reporter reduces imaging exposure times without the need for repeated sample excitation, which can result in cytotoxic artifacts.
NanoLuc® fusion proteins can be used in a variety of applications including: reporters of protein stability, probes for bioluminescent cell imaging (BLI) or as the donor signal in bioluminescent resonance energy transfer (BRET) applications for protein:protein or protein:small-molecule interaction studies. The brighter signal and blue-shifted emission spectrum from NanoLuc® luciferase result in less spectral overlap with fluorescent acceptors, resulting in better signal:background and dynamic range for BRET applications.
Additional NanoLuc® protein fusion vectors include the pFN31 and pFC32 Vectors. Generate N- or C-terminal Nluc fusion proteins using the Flexi® Vector Cloning System—a directional cloning method based on two rare-cutting restriction enzymes, SgfI and PmeI, that provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi® Vectors without the need to resequence.
Applications
Compound screening.
Protein:protein interactions.
Protein:small-molecule interactions.
Bioluminescent imaging.
Protein stability.
Reference
Hall, M.P. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chemical Biology 7(11) , 1848–57.
pNLF1-N [CMV/Hygro] Vector GenBank® Accession Number KF811457 and vector sequence text file .
pNLF1-C [CMV/Hygro] Vector GenBank® Accession Number KF811458 and vector sequence text file .
pNLF1-secN [CMV/Hygro] Vector GenBank® Accession Number KF811459 and vector sequence text file .
