A review by Ya-Li Liu and Zhan-Yun Guo, published this week in Amino Acids summarizes recent work of the authors and others using NanoLuc luciferase labeled protein/peptide hormones in receptor binding assays. Typically, studies assessing binding of hormones to receptors have used radioactive tracers. The brightness of NanoLuc luciferase makes bioluminescence an attractive alternative as a sensitive and safer option. Because cell membrane receptors are difficult to purify in quantity, the amounts available for experiments are usually limited. Therefore, tracers used in binding assays need to have a high affinity for the receptor, must not interfere with binding, and must be highly sensitive. Continue reading “Two light stories for Friday”
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.
For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.
Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.
The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.
The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:
Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.
You can read the Top 10 article in The Scientisthere.
Microbial cells outnumber the cells of our own bodies approximately 10:1, these microbes that live on our skin and along the epithelial linings of our internal tubes make up our microbiota*, and they can have major effects on our health. Most of our microbiota are commensal organisms, living in harmony with our body, but if you suppress our immune system or greatly reduce their populations with large doses of antibiotics, and you will soon see the effects of disrupting our microbiota.
There is much interest in the microbiota that inhabit our bodies. For instance several studies have indicated that intestinal microbes can play a big part in obesity, with changes in the makeup of the microbiota being a major risk factor (1). But many of these organisms are hard to learn about—the ones that inhabit the deep folds of our gut thrive in moist, warm, anaerobic conditions with lots of specialized nutrients, conditions that are very hard to replicate in the laboratory. For that reason, we don’t know much about many of the microbes that are the most abundant within us.
The Human Microbiome Project begun in 2008 by the National Institutes of Health (2) seeks to understand human microbiota and their relationship to human health. To do this, the researchers leading the project took a metagenomic approach—using advanced DNA sequencing technologies to sequence the genomes of human microbiota and get a look at the human microbiome—without culturing the microbes.
But to truly understand their biology, and to perhaps exploit what we learn to enhance human health we need to be able to manipulate these organisms. In particular, biologists who are interested in synthetic biology would like to use these micro-organisms to monitor what is going on in our bodies, particularly our guts. What better monitor for these hard-to-access places than an organism that is already well adapted to live there? Continue reading “Manipulating Microbiota: A Synthetic Biology Exploration of the Gut”
Robert 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.
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).
Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”
Here we continue our “month of little things” (#NanoLuc) with a deep sea angler fish (though in the video she doesn’t look so little). This female measures a mere 9cm long and uses a bioluminescent fishing lure (modified dorsal fin) to attract unsuspecting prey toward her gaping jaws. This video from the Monterey Bay Aquarium Research Institute is thought to be the first live footage of this species at this depth. Enjoy! In the deep, dark depths of the ocean it’s a little light that makes a difference.
This month we are celebrating a small thing—NanoLuc® luciferase, an enzyme whose tiny size and bright luminescence enable much more sensitive detection of intracellular events than other bioluminescent reporters. Scaling down the size of the luciferase protein makes it “fit” in situations where larger reporters do not, and also makes it less likely to interfere with natural biology than larger proteins. Scaling up the brightness allows you to detect the reporter at low abundance, enhancing sensitivity and allowing detection of small changes in gene expression at concentrations closer to physiological levels.
These properties of Nanoluc® luciferase allow its bioluminescence to be used to detect intracellular events in ways not possible before. One example of how this small size and intense brightness are being applied to help solve biological problems is the insertion of NanoLuc® luciferase into influenza viruses, where the genome size is small and does not tolerate large insertions. Unlike viruses incorporating larger luciferases, the influenza reporters incorporating NanoLuc® luciferase are stable, retain pathogenicity and are bright enough to track at low doses during the early stages of infection (1). You can find out more about the many applications of NanoLuc® luciferase here.
In the spirit of “celebrating small things” we will be sharing plenty of information about NanoLuc® applications over the next few weeks, and will also be highlighting other examples where “small” can be a beautiful thing. By definition, molecular biology is a study of the smallest building blocks of life, so it’s not hard to find an abundance of small things to talk about. We hope you will participate with us by commenting, liking or sharing posts, or by contributing your own ideas on little things that make a difference, or that just make you smile.
Genetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).
Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation
However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.
At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?
To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.
So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.