A Closer Look at C. difficile Biology with Luminescent Tagging

Clostridium difficile is a bacterium that infects around half a million people per year in the United States. The infection causes symptoms ranging from diarrhea to severe colitis, and it’s one of the most common infections contracted while staying in the hospital. As the global incidence of C. diff infection has risen over the past decade, so has the pressure to develop novel therapeutic strategies. This requires a thorough exploration of all aspects of C. difficile biology.

Two recent papers published by researchers at the University of Leiden have shed light on C. difficile physiology using HiBiT protein tagging. The HiBiT system allows detection of proteins in live cells using an 11 amino acid tag. The HiBiT tag binds to the complementary LgBiT polypeptide to reconstitute the luminescent NanoBiT® enzyme. The resulting luminescence is proportional to the amount of HiBiT-tagged protein that is present.

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NanoLuc® Luciferase: Brighter Days Ahead for In Vivo Imaging

nanoluc in vivo imaging

The development of NanoLuc® luciferase technology has provided researchers with new and better tools to study endogenous biology: how proteins behave in their native environments within cells and tissues. This small (~19kDa) luciferase enzyme, derived from the deep-sea shrimp Oplophorus gracilirostris, offers several advantages over firefly or Renilla luciferase. For an overview of NanoLuc® luciferase applications, see: NanoLuc® Luciferase Powers More than Reporter Assays.

The small size of NanoLuc® luciferase, as well the lack of a requirement for ATP to generate a bioluminescent signal, make it particularly attractive as a reporter for in vivo bioluminescent imaging, both in cells and live animals. Expression of a small reporter molecule as a fusion protein is less likely to interfere with the biological function of the target protein. NanoLuc® Binary Technology (NanoBiT®) takes this approach a step further by creating a complementation reporter system where one subunit is just 11 amino acids in length. This video explains how the high-affinity version of NanoBiT® complementation (HiBiT) works:

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CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics

Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.

Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.

In 2018, Promega R&D scientists published a paper in ACS Chemical Biology demonstrating the use of CRISPR/Cas9 to integrate the 11 amino acid, bioluminescent HiBiT tag directly into the genome to serve as an easily measured reporter for endogenous proteins. This provides a highly quantitative method for investigating cellular protein dynamics that sidesteps the need for cloning and other drawbacks to conventional methods, including the ability to measure changing protein dynamics in real-time. (For more details about CRISPR/Cas9 knock-in tagging and other applications, read this blog.)

While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.

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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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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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CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology

Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.

Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.

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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.

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Choosing a Tag for Your Protein

Flow diagram for purifying HaloTag fusion proteins
Overview of the HaloTag® Mammalian Protein Purification System.

You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.

Affinity Tags

The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains. Continue reading “Choosing a Tag for Your Protein”

All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.

With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA, like that induced by CRISPR-Cas9, is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”

Lighting Up GPCR Research with Bioluminescent Tagging

G Protein-Coupled Receptors (GPCRs) are a very large, diverse family of transmembrane receptors in eukaryotes. These receptors detect molecules outside the cell and activate internal signaling pathways by coupling with G proteins. Once a GPCR is activated, β-arrestins translocate to the cell membrane and bind to the occupied receptor, uncoupling it from G proteins and promoting its internalization.

Reporter tags are useful for studying the dynamics of GPCRs and associated proteins, but large tags can disrupt the receptors’ native functioning, and often overexpression of the tagged protein is required to obtain sufficient signal. Here is one example of how researchers have used the small, bright NanoLuc® luciferase to overcome these common challenges and answer questions about GPCRs. Continue reading “Lighting Up GPCR Research with Bioluminescent Tagging”