Tagged Target Protein Research Articles

Production of antimicrobial peptide arasin-like Sp in Escherichia coli via an ELP-intein self-cleavage system

Antibiotic resistance is a major public health threat to both humans and animals. There is an urgent need for antimicrobial agents with novel modes of action. Antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activity become the ideal alternative to traditional antibiotics. Here, the ELP-intein self-cleavage system was used to produce antimicrobial peptide arasin-likeSp in Escherichia coli. The tagged target protein (ELP-intein-arasin-likeSp) was mainly expressed in soluble, separated from cell lysates by the inverse transition cycling (ITC), and the arasin-likeSp was further purified by the self-cleavage of intein and the second round of ITC. The final yield of arasin-likeSp was about 3.56 mg/L. Purified arasin-likeSp exhibited significant antibacterial activities against the Gram-positive Bacillus subtilis and Gram-negative Vibrio harveyi bacteria. FE-SEM and PI staining analysis revealed that the arasin-likeSp treatment altered the morphology and membrane permeability of Bacillus subtilis and Vibrio harveyi. Collectively, these data suggest that arasin-likeSp is a candidate AMP for effective inhibition of Vibrio harveyi, a significant bacterial pathogen infecting marine fish and invertebrates. The ELP-intein self-cleavage system described here is a low-cost, simple and potential method for producing antimicrobial peptides, which lays foundations for the large-scale production of antimicrobial peptides in the future.

Development of BromoTag: A "Bump-and-Hole"-PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins.

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure–activity relationships of our bump-and-hole–PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo.

O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA.

The monosaccharide O-linked N-acetyl glucosamine (O-GlcNAc) is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA) fused to a nanobody. Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and nanobody-splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Target protein O-GlcNAcylation of JunB using nanobody-OGT Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody-splitOGA Alternate Protocol: Verification of the O-GlcNAc state of a tagged target protein through chemoenzymatic labeling Support Protocol: Cloning of new nanobody-OGT/nanobody-splitOGA and target protein pairs.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology.

In this work, the structuring of iron oxide nanoparticles via spray-drying (SD) of aqueous suspensions is investigated, leading to micrometer-sized aggregates with saturation magnetization comparable to that of the individual nanoparticles. Interestingly, the superparamagnetic behavior is retained despite the multicore structure. Modification of the aggregates via the addition of silica nanoparticles to the suspension allows for control of the resulting magnetization by adjusting the iron oxide content. Moreover, the morphology of the produced aggregates is gradually shifted from irregular inflated-like shapes in case of pure iron oxide aggregates to reach spherical structures when bringing the silica content to only 20%. The aggregates with different magnetization can be effectively separated in a simple column with an attached permanent magnet. Functionalization of pure iron oxide aggregates with a previously coupled ligand holding a nitrilotriacetic acid (NTA)-like moiety and subsequent loading with Ni2+ ions leads to the ability to bind 6-histidine (His6)-tagged target proteins via chelation complexes for magnetic separation. The application of the presented system for the purification of recombinant protein A in multiple cycles is shown. The recyclability of the separation system in combination with the high degree of magnetic separation is promising for future applications in the field of preparative in situ protein purification.

Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.

We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc.

An Automated High-Throughput Cell-Based Multiplexed Flow Cytometry Assay to Identify Novel Compounds to Target Candida albicans Virulence-Related Proteins

Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 °C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a "docking station peptide" into the hydrogel building block enables convenient incorporation of "docking protein"-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

Relationships between Cell Cycle Regulator Gene Copy Numbers and Protein Expression Levels in Schizosaccharomyces pombe

We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the “genetic tug-of-war” (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.

Recombinant tagging system using ribosomal frameshifting to monitor protein expression

For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis-elements of programmed -1 ribosomal frameshifting (-1RFS) as an embedded device. -1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a -1RFS element implanted between them, the unfused target and the target-GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing -1RFS signals. This limited-tagging system would be valuable for the real-time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large-scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products.

Cassia obtusifolia MetE as a Cytosolic Target for Potassium Isolespedezate, a Leaf-Opening Factor of Cassia plants: Target Exploration by a Compact Molecular-Probe Strategy

Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

Real-time on-line flow cytometry for bioprocess monitoring

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations ( R 2 ≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains ( P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.

The tandem affinity purification technology: an overview

Tandem affinity purification (TAP) is a methodology for the isolation of protein complexes from endogenous sources. It involves incorporation of a dual-affinity tag into the protein of interest and introduction of the construct into desired cell lines or organisms. Using the two affinity handles, the protein complex assembled under physiological conditions, which contains the tagged target protein and its interacting partners, can be isolated by a sequential purification scheme. Compared with single-step purification, TAP greatly reduces non-specific background and isolates protein complexes with higher purity. TAP-based protein retrieval plus mass spectrometry-based analysis has become a standard approach for identification and characterization of multi-protein complexes. The present article gives an overview of the TAP method, with a focus on its key feature-the dual-affinity tag. In addition, the application of this technology in various systems is briefly discussed.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain–intein tag for purification via a chitin–agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

Analysis of Protein−Small Molecule Interactions by Microscale Equilibrium Dialysis and Its Application As a Secondary Confirmation Method for on-Bead Screening

On-bead screening of one-bead one compound (OBOC) libraries is an ultra fast surface based primary high-throughput screening (HTS) method. Typically the binding of a tagged target protein to bead immobilized compounds or its altered enzymatic activity are detected. For an efficient and reliable ligand discovery process secondary assays to confirm on-bead compound activity in homogeneous solution are key to exclude artifacts and weak binders. Ideally they should allow to flag hit compounds with undesirable biophysical properties such as aggregation, unspecific binding, or insufficient solubility and the like. Here we demonstrate that miniaturized and parallelized equilibrium dialysis is an excellent and generic secondary confirmation method for hit compounds identified by on-bead screening. We further show that microscale dialysis can be reliably performed prior to decoding and resynthesis even with hit-compounds cleaved from the single beads. Down-scaling of the method takes advantage of the fluorescent tag, AIDA, which is integrated as permanent tracer in our library design. Our results suggest that microscale equilibrium dialysis followed by high performance liquid chromatography (HPLC) analysis is a generic, cheap, and meaningful confirmation method for identifying the most promising candidates within a series hit compounds derived from fluorescently tagged one-bead one-compound libraries.

Challenges and Rewards of Interaction Proteomics

The recent explosion of high throughput experimental technologies for characterizing protein interactions has generated large amounts of data describing interactions between thousands of proteins and producing genome scale views of protein assemblies. The systems level views afforded by these data hold great promise of leading to new knowledge but also involve many challenges. Deriving meaningful biological conclusions from these views crucially depends on our understanding of the approximation and biases that enter into deriving and interpreting the data. The challenges and rewards of interaction proteomics are reviewed here using as an example the latest comprehensive high throughput analyses of protein interactions in yeast.

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Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichia coli

The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SD-like sequences. Possible mechanisms behind the observed effects are discussed.

Expanded-bed adsorption immobilized-metal affinity chromatography

The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a process for production of recombinant biotherapeutics to standards of good manufacturing practice. It takes approximately 5 h to purify proteins from 10 liters of feedstock and a further 5-6 h to sterilize and regenerate the column.

Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

BackgroundAvailable in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.ResultsWe describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target.ConclusionSavings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.