Affinity Tag Research Articles

CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote.

Despite their importance to aquatic ecosystems, global carbon cycling, and sustainable bioindustries, the genomes of photosynthetic bacteria contain large numbers of uncharacterized genes. Here, we develop high-throughput endogenous fluorescent protein tagging in the cyanobacterium Synechococcus elongatus PCC 7942. From 400 targets, we successfully tag over 330 proteins corresponding to >10% of the proteome. We use this collection to determine subcellular localization, relative protein abundances, and protein-protein interaction networks, providing biological insights into diverse processes-from photosynthesis to cell division. We build a high-confidence protein-protein interaction map for the major components of photosynthesis, associating previously uncharacterized proteins with different complexes and processes. In response to light changes, we visualize, on second timescales, the reversible formation, growth, and fusion of puncta by two Calvin cycle proteins, suggesting that biomolecular condensation provides spatiotemporal control of the Calvin cycle in cyanobacteria. We envision that these insights, cell lines, and optimized methods will facilitate rapid advances in cyanobacteria biology and, more broadly, all photosynthetic life.

A pristine view of galactic globular clusters and their peripheries: Omega centauri

Abstract The central regions of the globular cluster Omega Centauri (ω Cen) have been extensively studied, but its outer regions and tidal structure have been less so. Gaia’s astrometry uncovered substantial tidal substructure associated with ω Cen, yet the lack of chemical tagging makes these associations tenuous. In this paper, we utilise the Gaia-Synthetic CaHK-band photometry, metallicities from the Pristine survey and Gaia’s astrometry to explore up to a clustercentric radius of 5 degrees from ω Cen. We identify ω Cen-like stars based on proper motion, colour-magnitude and colour-colour space, exploring the morphology, and stellar populations of the outer regions. Our probabilistic approach recovers the tidal tails of ω Cen, and we investigate the metallicity distribution of ω Cen ranging from a radius of 15 arcmin to the tidal radius, and beyond into the tidal tails. We present (1) two components between 15 arcmin and tidal radius at -1.83 and 1.45 dex which are also the dominant populations within 15 arcmin, and (2) the first evidence that the same two populations in the outer regions of the cluster are present outside the tidal radius and into the tidal tails. These populations are mixed about the stream, and are typically amongst the faintest stars in our sample; all indicating that the tidal tails are made of tidally stripped ω Cen stars.

Simplified MS2 phage-like particle production including a novel maturation protein internal fusion affinity tag

Phage-like particles (PLPs) are fabricated self-assembling nanoparticles derived from the structural elements of bacteriophages. These particles have biotechnological utility because of the ability to easily modify surface chemistry and compartmentalize nucleic acids or other materials. A consequential implementation of PLPs in diagnostics is as process controls in nucleic acid amplification tests, where control RNAs are packaged within the protein capsid and protected from degradation by RNases in the sample matrix. Key developments in PLP controls have enhanced the packing efficiency of RNAs into particles, reduced the complexity of their plasmid expression systems, and shifted purification from ultracentrifugation to affinity chromatography, producing progressively greater yields with higher purity. Expanding on prior improvements, this study establishes a revised set of plasmid vectors for Emesvirus zinderi (MS2) derived PLPs that streamline vector manipulations for rapid prototyping of new particles, provide validation of an alternative affinity tag for purification, and contributes a high-throughput low-volume spin column purification strategy. These advancements are combined with a novel internal fusion site in MS2 maturation protein A, a passive element of the MS2 capsid in prior PLP designs, that is capable of displaying polypeptides on the particles’ surface. The functionality of the chimeric maturation protein’s surface display is verified with an affinity tag fusion and subsequent purification. This advancement increases the number of available peptide display sites for the MS2 PLP platform with wide-ranging implications for future applications.

De novo design protein binders for MBP and GST tags.

Maltose-binding protein (MBP) and glutathione S-transferase (GST) are widely used solubility-enhancing protein tags, typically employed to address various issues related to protein expression and purification. The detection of these tags are usually achieved through binding of corresponding antibodies. Designing low-cost binders as alternatives to antibodies is of great significance. This study employed a de novo design approach, starting with a large number of protein scaffolds and screening out 6 candidate binders targeting MBP and 4 candidate binders targeting GST based on scoring functions. Flow cytometry low-affinity selection and biolayer interferometry (BLI) quantitative results showed that MBP and GST can interact strongly with one or several binders, exhibiting nanomolar binding. Among them, LZMB3 has a binding dissociation constant (KD) of 54.05±1.46nM, while LJGB3 and LJGB4 have KD values of 105.4±1.812nM and 437.9±17.69nM, respectively.

Improving soluble recombinant SARS-CoV-2 papain-like protease production in Escherichia coli through chaperonin and maltose-binding protein tag: purification and kinetic characterization

Although COVID-19 is now becoming endemic, SARS-CoV-2 persists potential jeopardy to clinically vulnerable populations. Hence, further study is still necessary to discover novel antiviral agents against SARS-CoV-2 for proactive preparedness. SARS-CoV-2 papain-like protease (PL Pro) is a target enzyme for searching anti-Covid candidates. Our prior study revealed the major formation of inclusion bodies during PL Pro expression in E. coli RIPL. In this study, we tried using chaperonin in the E. coli Arctic Express system and both codon optimization and maltose-binding protein (MBP) fusion protein to make PL Pro more soluble. Recombinant PL Pro encoded on the pET21d(+) plasmid was expressed in E. coli Arctic express. However, the soluble protein yield remained low and unstable due to suboptimal codon usage in the insert gene. Whereas, fusion of the MBP protein with optimized codon of PL Pro enhanced the enzyme expression and solubility. Recombinant PL Pro cleaved the linker between MBP and PL Pro, which served as a cleavage site recognized by PL Pro (LKGG↓A). The purified enzyme from a 200-mL culture generated 1 mL of pure PL Pro enzyme at a 1.913 mg/mL concentration. It exhibited favorable activity against the Z-RLRGG-AMC substrate, with a Km value of 33.40 μM and a Vmax of 5.10 RFU/min.

The dark side of fluorescent protein tagging-the impact of protein tags on biomolecular condensation.

Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used to study the formation and the properties of condensates in vitro and in vivo, but there is evidence that tags may perturb the condensation properties of proteins. In this study, we carefully assess the effects of protein tags on the yeast DEAD-box ATPase Dhh1, a central regulator of processing bodies (P-bodies), which are biomolecular condensates involved in mRNA metabolism. We show that fluorescent tags as well as a polyhistidine tag greatly affect Dhh1 condensation in vitro and lead to condensates with different dynamic properties. Tagging of Dhh1 with various fluorescent proteins in vivo alters the number of P-bodies upon glucose starvation and some tags even show constitutive P-bodies in nonstressed cells. These data raise concerns about the accuracy of tagged protein condensation experiments, highlighting the need for caution when interpreting the results.

The role of ubiquitination and deubiquitination in cancer lipid metabolism

The ubiquitin-proteasome system (UPS) is one of the main degradation systems within cells, catalyzing the tagging of proteins for degradation by ubiquitin molecules, which are then recognized and degraded by the proteasome. Lipid metabolism plays a crucial role in cellular energy metabolism and is closely associated with the occurrence and development of cancers. Recent research indicates that cancer lipid metabolism is regulated by intracellular proteins, including ubiquitination modifications. This review will explore the role of ubiquitination in regulating cancer lipid metabolism, summarize the latest research progress, and propose potential therapeutic strategies.

P-1369. Impact of the E152K Mutation in Resistance to Taniborbactam and Evolutionary Advantages of NDM-9

Abstract Background NDM is one of the most prevalent carbapenemases worldwide. Boronate-based inhibitors able to target Metallo−β-lactamases, MBLs, such as taniborbactam (TAN) and xeruborbactam (XER), are promising new drugs. However, NDM-9, the third most widespread variant of NDM-1 is refractory to the action of TAN. The E152K substitution in NDM-9 disrupts an attractive electrostatic interaction between the Glu residue in NDM-1 and the side chain of the inhibitor. We aimed to explore how the E152K was selected before the clinical use of TAN and its possible evolutionary pathways. The C-terminal dynamics that tags protein degradation is quenched in apo-NDM-9 The C-terminal dynamics that tags protein degradation is quenched in apo-NDM-9 Methods Minimum inhibitory concentrations of ceftazidime in Escherichia coli cells expressing NDM variants were determined in Luria Bertani (LB) media with increasing concentrations of dipicolinic acid (DPA). NDM-1 and NDM-9 were expressed in E. coli BL21(DE3) pLysS and purified by affinity chromatography. Site-directed mutagenesis was performed. Steady-state catalytic parameters were determined by initial rate measurements and/or by analysis of the progress curves, measured in HEPES buffer, pH 7.5 at 25°C. Deletion mutants in E. coli were obtained by CRISPR. NMR spectra were run in a 700 MHz Bruker spectrometer. Docking simulations were performed by using Glide.Figure 2.Protein levels in the periplasm of E. coli cells expressing different NDM variants as measured by immunoblotting upon zinc starvation. Results NDM-9 is able to confer higher resistance to ceftazidime than NDM-1 under zinc deprivation since apo-NDM-9 is more stable against the periplasmic proteases. NMR reveals that the C-terminal stretch recognized by the protease Prc is more rigid in apo-NDM-9, protecting this variant towards degradation. Despite this, E152K substitution is present only in NDM-9. We obtained new variants of more frequent substitutions such as M154L and A233V. The two double variants and the triple variant were generated. These combinations gave rise to NDM variant hyper-stable against degradation, which also showed resistance to TAN, as supported by docking experiments. Conclusion Resistance to TAN exhibited by NDM-9 is a secondary gain-of-function elicited by the E152K substitution, that represents a clinical threat since it stabilizes the protein under zinc limiting conditions. The finding of new combinations on the genetic background of NDM-9 giving resistance to TAN suggests that NDM-9 can further evolve by acquiring more mutations. Disclosures All Authors: No reported disclosures

Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori

Affinity tags are a crucial component in protein purification. Despite several indications that they can influence protein structure and function, this influence is often unknown or disregarded. This unnecessarily introduces ambiguity in the interpretation of in vitro data. To illustrate that, urea and ammonia yeast complementation assays are used as a screening tool to assess functional differences in various affinity tag positions, compared to the WT protein, using HpUreI, an acid‐gated urea channel of Helicobacter pylori. Yeast complementation assays test the pH‐dependent functionality of exogenous proteins expressed in deletion strains by observing growth. If the exogenous protein is able to replace the function of the deleted endogenous protein, yeast cells can demonstrate growth under specific assay conditions. The overall tag position and even a minor amount of residual N‐ or C‐terminal amino acids following tag cleavage exert a solute‐specific influence on HpUreI functionality, suggesting a complex solute selectivity mechanism and underscores the necessity for in vivo characterization. This cost‐effective yeast complementation assay can be adapted to test a broad range of solutes. It can be used as a preliminary screening tool for affinity tag positions or protein mutations before quantitative in vitro protein characterization.

Pre-folding purification procedures for inclusion body-derived non-tagged cationic recombinant proteins with multiple disulfide bonds for efficient refolding.

The production of disulfide-containing recombinant proteins often requires refolding of inclusion bodies before purification. A pre-refolding purification step is crucial for effective refolding because impurities in the inclusion bodies interfere with refolding and subsequent purification. This study presents a new pre-refolding procedure using a reversible S-cationization technique for protein solubilization and purification by reversed-phase high performance liquid chromatography. This pre-folding purification step improves refolding yield by effectively removing the refolding inhibitors from contaminates from bacterial inclusion bodies, and reducing proteolytically degraded products. Because this procedure does not require a peptide tag for affinity purification, it is a superior technique to subsequently perform a simplified downstream process wherein the affinity tag needs to be removed. This study reports improved refolding and purification procedure to obtain the highly cationic (pI = 9.25) mouse vascular endothelial cell growth factor (188 amino acids form) that is used as a model protein in our study; this protein shows a homodimeric conformation and possesses multiple disulfides.

Tools for Intersectional Optical and Chemical Tagging on Cell Surfaces.

We present versatile tools for intersectional optical and chemical tagging of live cells. Photocaged tetrazines serve as "photo-click" adapters between recognition groups on the cell surface and diverse chemical payloads. We describe two new functionalized photocaged tetrazine structures which add a light-gating step to three common cell-targeting chemical methods: HaloTag/chloroalkane labeling, nonspecific primary amine labeling, and antibody labeling. We demonstrate light-gated versions of these three techniques in live cultured cells. We then explore two applications: monitoring tissue flows on the surface of developing zebrafish embryos, and combinatorial multicolor labeling and sorting of optically defined groups of cells. Photoclick adapters add optical control to cell tagging schemes, with modularity in both tag and cell attachment chemistry.

Metabolic Tagging Technology of Exosomes- An Updated Review.

Exosomes are small extracellular vesicles secreted by various cell types, playing a crucial role in intercellular communication by carrying proteins, lipids, and nucleic acids, thus holding significant potential in diagnostics and therapeutics. Accurate labeling of exosomes is vital for studying their biogenesis, trafficking, and functional properties, enabling precise tracking and manipulation. This review examines current labeling techniques, including metabolic glycan labeling, chemical tagging, membrane fluorescent dyes, bio-conjugation, non-covalent labeling, and cell-engineering approaches. Each method is analyzed for its efficiency, specificity, and practicality, with attention to potential artifacts and challenges. Advancements in these techniques are essential for improving our understanding of exosome biology and developing exosome-based diagnostic and therapeutic strategies, providing researchers with valuable insights into state-of-the-art techniques and their applications in exosome research.

Cryo-electron tomography pipeline for plasma membranes

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells.

Mapping Dynamic Protein Clustering with AIEgen-Active Chemigenetic Probe.

Protein clustering/disassembling is a fundamental process in biomolecular condensates, playingcrucial roles in cell fate decision and cellular homeostasis. However, the inherent features of protein clustering, especially for its reversible behavior and subtle microenvironment variation, present significant hurdles in probe chemistry for tracking protein clustering dynamics. Herein, we report a bilateral-tailored chemigenetic probe, in which an "amphiphilic" AIEgenQMSO3Cl is covalently conjugated to a protein tag that is genetically fused to protein-of-interest (POI). Prior to target POI, the probeachieves a completely dark state in both aqueous environment and lipophilic organelles, ensuring an ultra-low background-interference. Upon reaching POI, the combination of synthetic molecule and genetically encoded protein allows for protein clustering-dependent ultra-sensitive response, with a substantial lighting-up fluorescence (67.5-fold) as protein transitions from disassembling to clustering state. Such ultra-high signal-to-noise ratio enables to monitor the dynamic and fate of IRE1clustering/disassembling under both acute and chronic endoplasmic reticulum (ER) stress. For the first time, we have demonstrated the use of chemigenetic probe to reveal therapy-induced ER stress and screen drugs inthree-dimensional scenario: microviscosity change, clustering dynamic, and cluster morphology. This chemigenetic probe design strategy would greatly facilitate the advancement of mapping protein dynamics in cell homeostasis and medicine research.

Mitochondrial Localization of Antioxidant Nanodrug Suppresses Ocular Inflammation by Alleviating Oxidative Stress on Cells.

External environments (e.g., pollutants, irritants, ultraviolet radiation, etc) probably activate oxidative stress on the ocular surface, further leading to inflammatory responses and cellular apoptosis. For treating ophthalmic diseases, one of the strategies is to regulate oxidative stress through antioxidants. Here we conjugate a polyphenolic antioxidant drug (i.e., caffeic acid) with a small peptide of protein tag to generate a peptide-drug conjugate as a nanodrug. With a self-assembled ability to form nanoparticles, the nanodrug mainly enters human corneal epithelial cells (HCEC) by caveolin-mediated endocytosis, succeeds lysosomal escape, and achieves mitochondrial localization of caffeic acid. Revealed by free radical scavenging experiments, the nanodrug shows considerable antioxidant capacities. In mouse leukemia cells of monocyte macrophage (RAW 264.7) induced by lipopolysaccharide (LPS), the nanodrug inhibits various pro-inflammatory cytokines (e.g., NO, IL-6, and TNF-α) by up-regulating the expression of anti-apoptotic protein (e.g., Bcl-2). As an investigation of alleviating oxidative stress by the mitochondrial localization of antioxidant, this work may establish an experimental foundation for the regulation of cellular redox balance, as well as provide different insights for the clinical development of antioxidant drug delivery systems in the treatment of ocular disease.

Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC50 of 17 nM and EphB4 with an IC50 of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC50 of 2.1 nM, suggesting that a DDR1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on non-kinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.

IP-to-MS: An Unbiased Workflow for Antigen Profiling.

Immunoprecipitation is among the most widely utilized methods in biomedical research, with applications that include the identification of antibody targets and associated proteins. The path to identifying these targets is not straightforward, however, and often requires the use of chemical cross-linking and/or gel electrophoresis to separate targets from an overabundance of immunoglobulin protein. Such experiments are labor intensive and often yield long lists of candidate antibody targets. Here, we describe an unbiased immunoprecipitation-to-mass spectrometry (IP-to-MS) method that relies on a novel protein tag to separate low abundance immunoprecipitated proteins from overwhelmingly abundant immunoglobulins. We demonstrate that the IP-to-MS serotyping workflow is highly reproducible and can be used for the identification of novel, patient-specific antigen targets in multiple disease states. Furthermore, we show that IP-to-MS may outperform conventional methods of antibody detection, including enzyme-linked immunosorbent assay, while also enabling patient stratification beyond what is possible with traditional approaches.

Protein Fusion of Biosynthetic Enzymes and a Thermo-Responsive Polypeptide Expedites Facile Access to Biocatalysts for Nucleotide Sugars.

Nucleotide sugars (NSs) are essential building blocks for the enzymatic assembly of glycans. In this study, we established a preparation and recycling avenue to the biocatalysts for the enzymatic synthesis of NSs. This approach involves fusing two enzymes into a bifunctional chimera and using elastin-like polypeptides (ET64) as a purification tag, which allows for easy recovery of these biocatalysts without the need for chromatography. We successfully constructed and obtained five bifunctional fusion enzymes (GalK-USP-ET64, GlmU-NahK-ET64, ManC-NahK-ET64, FKP-ET64, and NanA-CSS-ET64) for the synthesis of five common NSs (UDP-Gal, UDP-GlcNAc, GDP-Man, GDP-Fuc, and CMP-Neu5Ac). These enzymes were obtained using the Inverse Transition Cycling (ITC) process in yields ranging from 60 to 124 mg per liter of fermentation. The enzymatic synthesis of NSs was carried out on a scale from hundreds of milligrams to multiple grams using these biocatalysts. Furthermore, we investigated the reusability of these biocatalysts by recycling them from the reaction solution using the ITC process. The recycling of GalK-USP-ET64, GlmU-NahK-ET64, FKP-ET64, and NanA-CSS-ET64 was effectively achieved for 15, 13, 3, and 4 times, respectively. These biocatalysts could be used not only for the enzymatic synthesis of NSs but also for the chemoenzymatic synthesis of glycan biomolecules when coupled with glycosyltransferases.

A suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression.

Expression and purification of recombinant proteins in E. coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C-terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a completely scarless vector series with 9 different N-terminal tags. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.

A Structural Bioinformatics-Guided Study of Adenosine Triphosphate-Binding Cassette (ABC) Transporters and Their Substrates.

Adenosine triphosphate-binding cassette (ABC) transporters form a ubiquitous superfamily of integral membrane proteins involved in the translocation of substrates across membranes. Human ABC transporters are closely linked to the pathogenesis of diseases such as cancer, metabolic diseases, and Alzheimer's disease. In this study, four ABC transporters were chosen based on (I) their importance in humans and (II) their score in a structural bioinformatics screen aimed at the prediction of crystallisation propensity. The top-scoring ABC transporters' orthologs (Mus musculus-mouse ABCB5, Ailuropoda melanoleuca-giant panda ABCB6, Myotis lucifugus-little brown bat ABCG1 and Mus musculus ABCG4) were then expressed in Saccharomyces cerevisiae with a combined green fluorescent protein and polyhistidine tag, enabling visualisation and purification. After partial purification and in the presence of the detergent (n-dodecyl-β-D-maltoside), the kinetic parameters of the ATP hydrolysis reactions of the orthologs were determined, as well as the extent of stimulation of their activity when presented with putative substrates. We discuss the efficiency of such bioinformatics approaches and make suggestions for their improvement and wider application in membrane protein-structure determination.