Protein Analysis and Purification

Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases

Posted on by Ken Doyle
bet proteins brd nanoluc

Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.

One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).

Continue reading “Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases”

NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail

Posted on by Kyle Hooper

Recently, Gordon et al. published an atlas of protein:protein interactions of all proposed SARS-CoV-2 proteins expressed individually in HEK 293 cells (Table 1). The study tagged each of the viral proteins with an epitope tag and performed a pull-down of the expressed protein followed by trypsin digestion and mass spec analysis, a process referred to as affinity purification–mass spec analysis. The group identified 332 human proteins interacting with 27 SARS-CoV-2 proteins.

The interactions identified in the HEK 293 cells helped Appelberg et al. analyze interactions over time in SARS-CoV-2-infected Huh7 cells. Gordon et al. used the PPI data to identify FDA-approved drugs, drugs in clinical trials, and pre-clinical compounds that bound to the identified human proteins and labs in New York and Paris tested some of these drugs for antiviral effects.   

Table 1. The general functional area of human proteins identified to interact with individually expressed SARS-CoV-2 proteins as reported by Gordon, et al. (1). The SARS-CoV-2 proteins are classified as non-structural proteins (nsp#), structural proteins (E, M, and N) and accessory proteins (orf#).  
Continue reading “NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail”

NanoLuc® Luciferase Powers More than Reporter Assays

Posted on by Sara Klink
Bright NanoLuc® Luciferase

What can you do with a small, super bright luciferase? Amazing things. We’ve highlighted many of the papers and new applications that NanoLuc® luciferase has enabled on this blog. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating bioluminescent tools used to study endogeneous protein dynamics, target engagement, protein degradation, immunodetection and more. So where did the NanoLuc luciferase come from and how does one enzyme power so many research capabilities? Read further for a primer on the various technologies and applications developed from this enzyme over the last 10 years.

Continue reading “NanoLuc® Luciferase Powers More than Reporter Assays”

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

Posted on by Natalie Larsen

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).

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

Giant Rodent, Lowered Cancer Rates: What Genetic Analysis Reveals about the Capybara and Cancer

Posted on by Kari Kenefick

Do you find the thought of a giant rodent off-putting? Do your thoughts go to huge rats running amuck in dark allies, threatening unsuspecting passers by?

I personally hold rodents in low esteem. Rats, mice…who needs them? With the exception of cavies. I spent countless hours as a child playing with guinea pigs. We had as many as 16 of these little rodents at one time (the males are very capable of chewing or climbing out of cardboard boxes to reach a female in the next box). The baby guinea pigs were very cute and the adults had quite pronounced personalities, and a lot of attitude.

It was this history with guinea pigs that made me interested in learning more about the largest rodent in the world, the South American capybara (Hydrochoerus hydrochaeris). These family-oriented herbivores are found in savannas and forested areas, living in groups of as many as 100 members. They are excellent swimmers and can remain underwater for as long as 5 minutes. In fact, capybara mate only in the water. (Perhaps it’s not surprising then that the South American alligator, the caiman, is one of the capybara’s greatest predators.)

Photos of an adult male capybara.
A male capybara, with a scent gland (called a morillo) on his head. Photo by: Charles J Sharp – Own work, from Sharp Photography, sharpphotography, CC BY-SA 4.0.

With their squared-off nose and lack of tail, capybaras actually resemble guinea pigs. However, these oversized cavies weigh as much as 40 pounds. and can reach 24” at the shoulder, the size of an average standard poodle. Guinea pigs, on the other hand, weigh in at 2–3 pounds, and are 3–4” tall.

Their proportions make capybaras 60 times more massive than their closest relatives, rock cavies (Kerodon sp.) and 2,000 times more massive than the common mouse (Mus musculus). This tremendous size difference is why Herrera-Álvarez et al. took a closer look at the capybara, studying its propensity to develop cancer and other tradeoffs that would seem to coincide with its exceptional size.

Continue reading “Giant Rodent, Lowered Cancer Rates: What Genetic Analysis Reveals about the Capybara and Cancer”

Targeted Protein Degradation: A Bright Future for Drug Discovery

Posted on by Ken Doyle
targeted protein degradation and protacs

Our cells have evolved multiple mechanisms for “taking out the trash”—breaking down and disposing of cellular components that are defective, damaged or no longer required. Within a cell, these processes are balanced by the synthesis of new components, so that DNA, RNA and proteins are constantly undergoing turnover.

Proteins are degraded by two major components of the cellular machinery. The discovery of the lysosome in the mid-1950s provided considerable insight into the first of these degradation mechanisms for extracellular and cytosolic proteins. Over the next several decades, details of a second protein degradation mechanism emerged: the ubiquitin-proteasome system (UPS). Ubiquitin is a small, highly conserved polypeptide that is used to selectively tag proteins for degradation within the cell. Multiple ubiquitin tags are generally attached to a single targeted protein. This ill-fated, ubiquitinated protein is then recognized by the proteasome, a large protein complex with proteolytic activity. Ubiquitination is a multistep process, involving several specialized enzymes. The final step in the process is mediated by a family of ubiquitin ligases, known as E3.

Continue reading “Targeted Protein Degradation: A Bright Future for Drug Discovery”

NanoLuc: Tiny Tag with a Big Impact

Posted on by Darcia Schweitzer

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”

Choosing a Tag for Your Protein

Posted on by Sara Klink

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.

Immunofluorescent detection of HiBiT-tagged proteins in CRISPR-edited cell pools and clones using the Anti-HiBiT Monoclonal Antibody.
CRISPR-Cas9 editing knocked-in HiBiT at the endogenous locus of proteins with varying subcellular localization. Fixed CRISPR-modified clones or pools of cells were imaged by immunofluorescent staining using the Anti-HiBiT Monoclonal Antibody (red) and Hoechst dye (blue). Panel A. VCL-HiBiT pool. Panel B. SMARCA4-HiBiT clone. Panel C. HDAC2-HiBiT clone. Panel D. HSP90B1-HiBiT pool.

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”

Looking Back: Cell-Free Expression Systems Helped to Characterize Proteins Involved in Hypoxia Response

Posted on by Gary Kobs
Structur of a HIF-1a-pVHL-ElonginB-ElonginC complex
Structure of a HIF-1a-pVHL-ElonginB-ElonginC complex

William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza were awarded the 2019 Nobel Prize in Physiology or Medicine for their discoveries of how cells sense and adapt to oxygen availability.

Kaelin and Ratcliffe’s labs focused their efforts on the transcription factor HIF (hypoxia-inducible factor). This transcription factor is critical in the cellular adaptation of to changes in oxygen availability.

When oxygen levels are elevated cells contain very little HIF. Ubiquitin is added to the HIF protein via the VHL complex and it is degraded in the proteasome.  When oxygen levels are low (hypoxia) the amount of HIF increases.

In 2001 both groups published articles characterizing the interaction between VHL and HIF, and these articles were referenced by the Nobel Prize Organization in their press release about this year’s award. (1,2). Both studies demonstrated that under the normal oxygen conditions hydroxylation of proline residue P564 enabled VHL to recognize and bind to HIF.

The use of cell free expression (i.e., TNT Coupled Transcription/Translation System) by both labs was key in the characterization of the VHL:HIF interaction The labs utilized HIF and VHL 35-S labeled proteins generated via the TNT system under both normal or in a hypoxic work station to:

  • Determine the affect of ferrous chloride and cobaltous chloride on the interaction
  • Map the specific region of HIF required for the interaction to occur (556-574)
  • Determine the effect of HIF point mutations on the interaction
  • Use synthetic peptides to block the interaction
  • Conclude that a factor in mammalian cells was necessary for the interaction to occur.

Literature Cited

  1. Ivan, M et al. (2001) HIF Targeted for VHL-Mediated Destruction by Proline Hydroxylation: Implications for O2 Sensing. Science 292: 464–67.
  2. Jaakkola, P. et al. (2001) Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation of Complex by O2– Regulated Prolyl Hydroxylation. Science 202, 468–72 .

Related Posts

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

Posted on by Sara Klink

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”