NanoLuc® Luciferase Powers More than Reporter Assays

Bright NanoLuc® Luciferase

NanoLuc® luciferase has been discussed many times on this blog and our web site because the enzyme is integral to studying genetic responses and protein dynamics. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating tools used to study endogeneous protein interactions, target engagement, protein degradation and more. So where did the NanoLuc® luciferase come from and how does a one enzyme power several technologies?

The story of NanoLuc® luciferase starts with a deep sea shrimp and its bioluminescent enzyme that evolved with some tinkering from Promega scientists. The result: A small 19kDa reporter enzyme with a luminescent signal 100X brighter than that of firefly or Renilla luciferase. The signal intensity of NanoLuc® luciferase provides a sensitive reporter that can be used to investigate low-level protein expression and endogenous proteins. Furthermore, the small size of the engineered NanoLuc® luciferase means the enzyme can be used to create reporter viruses without disrupting packaging or measure endogenous expression using CRISPR-Cas9 gene editing. Read how NanoLuc® luciferase was developed in this article.

With such a bright enzyme, NanoLuc® luciferase would be an ideal luminescent donor for bioluminescence resonance energy transfer (BRET) assays that measure molecular proximity. We took it a step further and partnered NanoLuc® luciferase with the HaloTag® protein and its fluorescent ligands to create the NanoBRET™ assay. The bright, blue-shifted signal from the NanoLuc® donor combined with the far-red-shifted HaloTag® acceptor created a protein interaction assay with optimal spectral overlap, increased signal, and lower background compared to conventional BRET assays. This article describes how the live-cell NanoBRET™ assays were developed.

Not only can you measure protein:protein interactions, but NanoBRET™ technology powers the live-cell target engagement (TE) assays that measure how tightly a compound binds to a protein (compound affinity) and how much compound binds to the protein (target protein occupancy). You can even assess how long a protein binds to the target protein (residence time) under physiological conditions. Read how NanoBRET™ technology measures target engagement in live cells.

While a BRET-based assay is one way to assess how proteins interact, another method involves using two subunits with low binding affinity that structurally complement each other to produce a bright luminescent enzyme. The NanoLuc® Binary Technology or NanoBiT is composed of Large BiT (LgBiT), an 18kDa protein, and Small BiT (SmBiT), a 1.3kDa peptide. The two subunits only form an enzyme when fused to target proteins and the two target proteins interact, bringing SmBiT and LgBiT together. Learn how NanoBiT was developed in this ACS Chemical Biology article.

While NanoLuc® luciferase may be a small reporter enzyme, having a small peptide only 11 amino acids would be useful for measuring endogenous protein abundance. The high affinity binding between the peptide HiBiT and LgBiT produces a bright luminescent signal in the presence of substrate. Just tag the protein of interest by inserting HiBiT into the endogenous locus using CRISPR-Cas9 gene editing, add a reagent containing LgBiT and substrate, and measure the luminescence. The HiBiT tag offers a quantitative and sensitive method for investigating protein abundances and dynamics. Read about using CRISPR with the HiBiT luminescent peptide.

NanoLuc® Luciferase enzyme

Want to learn more about NanoLuc® luciferase and its capabilities? Check out our NanoLuc® Luciferase technology page for additional resources and applications.

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Sara Klink

Technical Writer at Promega Corporation
Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

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