Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository

Researchers having been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”.  One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes this so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information.  Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc).  The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1).  From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase.  This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction.  NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction.  NLuc lets you work at physiological concentrations.  NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in.  NLuc is very inviting for endogenous tagging as it is only 19kDa.  An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).

Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading

Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal. Continue reading

About the Development of an Improved BRET Assay: NanoBRET

"Protein BRD4 PDB 2oss" by Emw - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:Protein_BRD4_PDB_2oss.png#/media/File:Protein_BRD4_PDB_2oss.png

“Protein BRD4 PDB 2oss” by Emw – Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons – https://commons.wikimedia.org/wiki/File:Protein_BRD4_PDB_2oss.png#/media/File:Protein_BRD4_PDB_2oss.png

One of the more exciting reporter molecules technologies available came online in the past year, with the launch of the Promega NanoBRET™ technology. While it’s easy for me, a science writer at Promega, to brag, seriously, this is a very cool protein interactions tool.

A few of the challenges facing protein-protein interactions researchers include:

  • The ability to quantitatively characterize protein-protein interactions
  • Ability to examine protein-protein interactions in situ, in the context of the living cell

A goal of the NanoBRET™ developers was to improve the sensitivity and dynamic range of traditional BRET technology, in order to address these challenges.

In May 2015 these researchers published an article outlining their efforts to create NanoBRET technology in ACS Chemical Biology, in an article entitled, “NanoBRET—A Novel BRET Platform for the Analysis of Protein-Protein Interactions”. Here is a brief look at their work.

Continue reading

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

14231183 WB MS Protein Interactions Hero Image 600x214Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring these fluid interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal. Continue reading

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading

Uncovering Protein Autoinhibition Using NanoBRET™ Technology

13305818-protein ligandIntroducing new assays or technologies is meant to make it easier for you to perform research and craft experiments to test hypotheses. However, scientists are creative people, and new technologies or assays may just be the catalyst for a crucial experiment or new data you are seeking. In the case of a recent Proceedings of the National Academy of Sciences USA article, Wang et al. used the principle of our NanoBRET™ assay to understand how ERK1/2 phosphorylation of Rabin8, a guanine nucleotide exchange factor, influenced its configuration and subsequent activation of Rab8, a protein that regulates exocytosis. Continue reading

Using an Improved Bioluminescence Resonance Energy Transfer Technology to Monitor Bromodomain Histone Interactions

NanoBRET™ TechnologyTraditional techniques for studying protein:protein interactions (e.g., affinity capture or mass spectrometry) are limited in that they present a static picture of what is a dynamic process. To gain a more accurate and complete picture of interactions, they need to be studied n the context of the cellular environment within which they occur. In the webinar: Monitoring Protein:Protein Interactions in Living Cells Using NanoBRET™ Technology, Dr. Danette Daniels presented an improved Bioluminescence Resonance Energy Transfer (BRET) Technology that was developed to specifically study dynamic interactions in living cells. The NanoBRET™ Technology combines the small, extremely bright NanoLuc® Luciferase as the donor with a fluorescently labeled HaloTag® protein fusion tag as the acceptor to form a BRET assay. This assay offers an increased signal and decreased spectral overlap compared to other BRET technologies. Among other things, these features provide a larger signal range and the brightness of the NanoLuc® Luciferase enables protein:protein interaction analysis even with the donor protein expressed at low levels. Continue reading

Tools for Observing Protein Behaviors Inside Cells

nanoluchomepageimageIn this BioCompare video, Promega scientist, Dr. Keith Wood, explains how the smaller, brighter and sensitive NanoLuc® Luciferase allows scientists to observe protein behaviors inside cells, including rare events. See how a luciferase can now be used to investigate protein activities as well as for traditional luciferase genetic reporter assays.

http://www.biocompare.com/Life-Science-Videos/156795-Tools-for-Analysis-of-Protein-Behavior-In-Living-Cells/?arev=true

A Bright Future with a Small, Versatile Luciferase

Promega Connections is pleased to present this guest blog from Kyle Hooper, PhD, Cellular Analysis Specialist in the North America Group at Promega Corporation

NanoLuc™ Luciferase has properties that make it especially useful for imaging. Here a beta-2 adrenergic receptor/NLuc fusion is directed to the cell surface.

Check out the new webinar about NanoLuc™ Luciferase just posted to the Promega Webinars site. NanoLuc™ Luciferase is derived from a deep sea shrimp and is ~100-fold brighter than either firefly or Renilla luciferase. Continue reading