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?

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From Live Cells to Lysates: Adapting NanoBiT to a Biochemical Assay Format

The ability to target protein interactions with low solubility or weak binding affinities can present a significant challenge when it comes to drug screening. The beauty of these types of challenges we often face in the lab is that finding solutions to these problems doesn’t necessarily require brand new tools. Sometimes we already have the right tools in our arsenal and, with just a little creativity and collaboration, they can be adapted to address the challenge at hand.

In the following video, Dr. Mohamed (Soly) Ismail, a Postdoctoral Fellow at the Downward Lab of the Francis Crick Institute, presents the perfect example of this with his novel approach to the NanoBiT® Protein:Protein Interaction Assay. Through a collaboration with Promega R&D Scientists, Dr. Ismail has translated the assay into a cell-free, biochemical format, termed the NanoBiT Biochemical Assay (NBBA).

Watch the subtitled version of the video here >

Continue reading “From Live Cells to Lysates: Adapting NanoBiT to a Biochemical Assay Format”

Popular Papers from Promega Authors

Promega is a chemistry and instrument supplier to scientists in diverse industries and research labs around the world. True. But we are more than just a supply company; we are scientists dedicated to supporting the work of other scientists. We want the science behind the technologies we develop to be both vetted and valued by the scientific community at large, which is one reason our scientists take the time to prepare and submit manuscripts to peer-reviewed journals. Here we call out some of our published research papers that were highly read in 2019. In the journal ACS Chemical Biology alone, five Promega-authored papers were among the top 10 most read papers in 2019. Here’s a quick review of the highlights from these ACS papers.

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NanoLuc: Tiny Tag with a Big Impact

Synthetic biology—genetically engineering an organism to do or make something useful—is the central goal of the iGEM competition each year. After teams conquer the challenge of cloning their gene, the next hurdle is demonstrating that the engineered gene is expressing the desired protein (and possibly quantifying the level of expression), which they may do using a reporter gene.

Reporters can also play a more significant role in iGEM projects when teams design their organism with reporter genes to detect and signal the presence of specific molecules, like environmental toxins or biomarkers. Three of the iGEM teams Promega sponsored this year opted to incorporate some version of NanoLuc® Luciferase into their projects.

NanoLuc® luciferase is a small monomeric enzyme (19.1kDa, 171 amino acids) based on the luciferase from the deep sea shrimp Oplophorus gracilirostris. This engineered enzyme uses a novel substrate, furimazine, to produce high-intensity, glow-type luminescence in an ATP-independent reaction. Unlike other molecules for tagging and detecting proteins, NanoLuc® luciferase is less likely to interfere with enzyme activity and affect protein production due to its small size.

NanoLuc® Luciferase has also been engineered into a structural complementation reporter system, NanoBiT® Luciferase, that contains a Large subunit (LgBiT) and two small subunit options: low affinity SmBiT and high affinity HiBiT. Together, these NanoLuc® technologies provide a bioluminescent toolbox that was used by the iGEM teams to address a diverse set of biological challenges.

Here is an overview of each team’s project and how they incorporated NanoLuc® technology.

Continue reading “NanoLuc: Tiny Tag with a Big Impact”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. Continue reading “Top 5 Most Read Promega Papers in 2017”

Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology

Cannabinoids. What are they? Sometimes, Wikipedia can give a nice definition:

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons
Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

A cannabinoid is one of a class of diverse chemical compounds that acts on cannabinoid receptors in cells that alter neurotransmitter release in the brain. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals), the phytocannabinoids (found in Cannabis and some other plants), and synthetic cannabinoids (manufactured artificially).

Synthetic cannabinoids (SCs) were originally created for the scientific investigation of two cannabinoid receptors, CB1 and CB2, but have made their way to the streets as “safe” and “legal” alternatives to marijuana.

The problem is that these SCs engage the cannabinoid receptors more completely and with higher affinity than anything derived from marijuana. As a result, SCs can produce serious side effects that often require medical attention. In fact, you are 30 times more likely to seek emergency medical attention following the use of an SC than with natural cannabinoid sources like marijuana. Continue reading “Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology”

For Protein Complementation Assays, Design is Everything

Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use  a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.

A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.

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Studying Mitochondrial Fission with NanoBiT Complementation Assay

Vote for NanoBiT™ in the Scientist's Choice Awards and get a chance to win a $500.00 US Amazon voucher from Select Science.
Vote for NanoBiT™ in the Scientist’s Choice Awards and get a chance to win a $500.00 US Amazon voucher from Select Science.

Motivation
It’s a new year. Whether you’re a self-improvement fanatic or just ready for good things to start happening, you’ve got a plan. You might be changing up an old exercise routine or trying a new cooking technique.

And at work, you are digging deeper; this is the year you illuminate the protein interactions that you’ve previously not been able to visualize.

Good news. There is a new protein complementation assay that can help. Studying-mitochondrial-fission-poster

About NanoBiT
NanoBiT™ Complementation Reporter is a recently developed protein interaction assay that features the improved NanoLuc® luciferase. NanoLuc, originally isolated from a deep sea shrimp, is a small luciferase that provides a much brighter signal than firefly luciferase.

About Split Luciferase Systems
If you’re interrogating two proteins to understand the conditions under which they interact, a split luciferase system enables you to tag each protein with a luciferase subunit. Interaction of the tagged proteins facilitates the complementation of the subunits, resulting in a luminescent signal. Continue reading “Studying Mitochondrial Fission with NanoBiT Complementation Assay”

Key Advances in PPI Research

small linkedin thumbnailOur understanding of the microscopic world has been shaped by the tools available to monitor and visualize cellular interactions. We “stand on the shoulders of giants” to propel our research to even greater heights. Studying protein-protein interactions (PPI) has proved fruitful for our understanding of cellular metabolism, signal transduction, and more. Scientists are starting to build whole organism interactomes (kindred to the metabolome and genome) that could have huge implications towards understanding and treating disease. Let us take a trip down memory lane to see where we have come from.  Continue reading “Key Advances in PPI Research”