Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

In E. coli and other prokaryotes such as Staphylococcus aureus, and Mycobacterium tuberculosis, biotin protein ligase (BirA) is an emerging drug target as it is the sole enzyme capable of biotin transfer onto the BCCP subunit of ACC. There is currently a gap in simple yet efficient assays for rapidly identifying and characterising inhibitors targeting BirA. We present for the first time the development and validation of a simple and reliable DSF-GTP assay for the high-throughput screening of BirA: ligand interactions using a new GFP-tagged BirA of E. coli. In addition, we developed a new GFP-based biotinylation activity assay taking advantage of a GFP tethered with an AviTag. The data obtained with these assays revealed new insights into how the binding of individual or combinations of ligands affect the overall thermal stability and affinity of BirA. The DSF-GTP assay has a Z' value of 0.785 that makes it an excellent tool for future high-throughput screening of inhibitory compounds

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure–activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Samuel Askin and Taylor Wilkerson Interview, June 16, 2020

Twitch streamers Samuel Askin and Taylor Wilkerson briefly recount their experiences growing up prior to the COVID-19 pandemic. Wilkerson recalls moving from state-to-state while Askin discusses living in Helena, MT and then moving to Texas with Taylor before settling back in Helena. Askin and Wilkerson both describe the video games they played throughout their lives and note that they first met over an online game. The couple discuss their experiences using Twitch to stream their gaming sessions online and their preference for cooperative games. Askin and Wilkerson describe living in Helena during COVID-19. They note that the pandemic did not dramatically alter their work because they worked from home before the pandemic but mention difficulties such as being away from family and avoiding anxiety over COVID-19 news. The couple discuss their opinions on the state of Montana reopening and the increase of COVID-19 cases in June. Askin and Wilkerson also recount their experiences at the Black Lives Matter protests in Helena.https://scholarworks.umt.edu/universityofmontanacovid19_oralhistory/1000/thumbnail.jp

A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices

Protein detection in complex biological fluids and matrices has become a widely diversified field utilizing a number of different technologies. The quantification of target proteins in complex media such as serum remains a challenge for most technologies such as mass spectrometry, ELISA and western blot. Quantitative Immuno-PCR has been heavily used for antigen detection in immunoassays, but minimally so for quantifying affinity-tagged proteins expressed or circulating in complex matrices - despite its high sensitivity and robustness - because it suffers from detrimental background effects arising from its extreme detection power. We report the development of a universal qIPCR-based platform for the reproducible detection of dual affinity-tagged protein analytes in crude complex matrices such as serum and cell culture media or lysates. The system uses a couple of high-affinity antibodies against two affinity tags (GFP and HA) for the detection of dual-tagged proteins. The dual-tagged analyte is immuno-captured by one of its tags, while the second tag is bound by a detection device consisting of a new kind of self-assembled antibody-DNA conjugate. The new qIPCR platform enabled picomolar quantification of dual-tagged sortase in crude serum in 4 h including the PCR step

IgG-detection devices for the Tus-Ter-lock immuno-PCR diagnostic platform

The number of new Immuno-PCR technologies and applications is steadily growing as a result of a general need for more sensitive immunoassays for early detection of diseases. Although Immuno-PCR has been demonstrated to be superior to its immunoassay counterpart, it is still regarded as a challenging technology due to various problems arising from its increased detection power, such as high background noise as well as substantial batch-to-batch reproducibility issues. Current efforts have intensified to produce homogeneous universal protein-DNA conjugates to simplify this technology and render it more robust. We have recently developed a new quantitative Immuno-PCR (qIPCR) technology using the Tus-Ter-lock (TT-lock) interaction to produce homogeneous protein-DNA conjugates that can detect very small numbers of disease-related antibodies. We now report the further development of the TT-lock Immuno-PCR platform for the quasi universal quantitative detection of antigens and mammalian IgG. For this, Tus was fused to various IgG-binding proteins -i.e. protein G, protein L and their LG chimera - and self-assembled to the TT-lock-T template. These detection devices were then evaluated and applied in various direct and indirect Immuno-PCR formats. The direct TT-lock qIPCR could detect goat anti-GFP IgG at concentrations as low as 0.3 pM and total human IgG in serum samples with great sensitivity. Further indirect TT-lock qIPCR systems were developed that could detect 1 pM of GFP and 10 pM of measles nucleoprotein. In all cases, the superiority of the TT-lock Immuno-PCR was demonstrated in terms of sensitivity over an analogous Protein G-Peroxidase ELISA

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader

In-gel detection of biotin–protein conjugates with a green fluorescent streptavidin probe

Exploitation of the (strept)avidin–biotin interaction is extremely valuable in a variety of biotechnological applications. Biotin is often covalently linked to proteins or nucleic acids. Determination of the degree of biotinylation of such macromolecules is essential for downstream applications. There is currently a gap in simple yet efficient assays for rapidly quantitating protein biotinylation, as staple methods may produce unclear results or rely on immuno- or competitive assays. We present a simple and reliable electrophoretic method to determine the relative extent of biotinylation of macromolecules. The method relies on complex formation between a biotinylated macromolecule and a streptavidin probe resulting in an electrophoretic mobility shift of the complex detectable by SDS-PAGE. Finally, a green fluorescent protein labelled streptavidin probe was developed to eliminate the need for staining and reduce assay time

Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

The development of differential scanning fluorimetry and the high-throughput capability of Thermofluor have vastly facilitated the screening of crystallization conditions of proteins and large mutant libraries in structural genomics programs, as well as ligands in drug discovery and functional genomics programs. These techniques are limited by their requirement for both highly purified proteins and solvatochromic dyes, fueling the need for more robust technologies that can be used with crude protein samples. Here, we present the development of a new high-throughput technology for the quantitative determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. This technology is based on the principle that a change in the proximal environment of GFP, such as unfolding and aggregation of the protein of interest, is measurable through its effect on the fluorescence of the fluorophore. Protein stability data was generated for twelve GFP-tagged proteins including monomeric and multimeric, DNA-binding, RNA-binding, proteolytic, heat-shock and metabolic proteins of Escherichia coli, Burkholderia pseudomallei, Staphylococcus aureus, dengue and influenza (H5N1) viruses. The technology is simple, fast and insensitive to variations in sample volumes, and the useful temperature and pH range is 30–80 °C and 5–11 respectively. The system does not require solvatochromic dyes, reducing the risk of interference. The protein samples are simply mixed with the test conditions in a 96-well plate and subjected to a melt-curve protocol using a real-time thermal cycler. The data are obtained within 1–2 h and include unique quality control measures