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Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5' end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations., Püllmann et al developed a modular Golden Gate-based secretion system, which enabled production and one-step purification of active fungal unspecific peroxygenases (UPOs) in yeast. Their system was applied to an enantioselective conversion on a preparative scale and may be used in the future for other genes of interest that are suitable for production in yeast.

Macropinocytosis is among ubiquitous cellular uptake mechanisms of peptide-based intracellular delivery. Due to its capability of engulfing large macromolecules, macropinocytosis shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. We previously reported SN21, a peptide derived from the N-terminus of stromal cell-derived growth factor 1α (SDF-1α), as a potent macropinocytosis inducer. In this work, we obtained the 8-residue analog P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A with membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

We report an accurate method to determine DNA barcodes from the dwell time measurement of protein tags (barcodes) along the DNA backbone using Brownian dynamics simulation of a model DNA and use a recursive theoretical scheme which improves the measurements to almost 100% accuracy. The heavier protein tags along the DNA backbone introduce a large speed variation in the chain that can be understood using the idea of non-equilibrium tension propagation theory. However, from an initial rough characterization of velocities into “fast” (nucleotides) and “slow” (protein tags) domains, we introduce a physically motivated interpolation scheme that enables us to determine the barcode velocities rather accurately. Our theoretical analysis of the motion of the DNA through a cylindrical nanopore opens up the possibility of its experimental realization and carries over to multi-nanopore devices used for barcoding., We report a method for DNA barcoding from the dwell time measurement of protein tags (barcodes) along the DNA backbone using Brownian dynamics simulation of a model DNA and use a recursive scheme to improve the measurements to almost 100% accuracy.

The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems., This minireview describes the development of NIR chemical probes for various protein-tag systems.

The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.

Silicatein is an enzyme capable of catalyzing silica formation under mild conditions and is a promising catalyst for the fabrication of biohybrid materials. However, unfavorable aggregation of silicatein makes it unsuitable for use in material fabrication. In this study, a soluble protein tag (ProS2) and a carbohydrate-binding module (CBM) were used to develop a soluble and cellulose-binding fusion silicatein, ProS2-Sil-CBM, which can be efficiently immobilized on cellulose to form silica on it. ProS2-Sil-CBM was soluble in aqueous media and strongly bound to cellulose. ProS2-Sil-CBM bound on cellulose catalyzed the formation of a silica layer on the cellulose in the presence of tetraethyl orthosilicate as the substrate. Scanning electron microscopy (SEM) and surface elemental analysis confirmed the formation of silica on cellulose. This technique can be used to fabricate inorganic-organic hybrid materials to immobilize biomolecules and can be applied to develop novel biocatalytic systems, biosensors, and tissue culture scaffolds.

Protein is the basis of all biological processes. Protein studies, especially the discovery of protein functional regulation network in life activities, have become an important scientific issue that has drawn increasing attention from scientists after the Human Genome Project. However, large-scale protein research needs an efficient and standard platform. Building a resource platform of model organism where every protein is tagged at organismal level is the key to solving this problem. However, although genome-wide protein labelling has been achieved in Saccharomyces cerevisiae , Caenorhabditis elegans and Drosophila , genome-wide protein tagging in mice is still very challenging. Genome tagging project (GTP) in mice has been proposed based on two state-of-art biotechnologies, i.e., androgenetic haploid embryonic stem cell (so-called “artificial spermatid”)-mediated semi-cloning technology and CRISPR-Cas9-based genome-editing technology. GTP involves two steps: first, the “artificial spermatid” lines carrying a tagged protein are constructed in vitro by endogenously inserting a tag sequence into a specific protein-coding gene via CRISPR-Cas9; second, the tagged “artificial spermatids” can be injected into oocytes to obtain tagged mice in one step. This strategy by combining “artificial spermatids” and CRISPR-Cas9 is simple, efficient, and low-cost, and thus can be used to generate tagged mice at the genome-wide scale. The goal of GTP is to tag every protein in mice and, we believe, the implementation of GTP will promote to investigate the protein expression, localization and function in vivo through standardized tag antibody-based assays, which will greatly enhance the advances of life sciences.

Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8×10(4) M(−1)s(−1) with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino aids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for sub-stochiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli., This work describes the production of bioactive human growth hormone tagged with the fusion protein PelB‐SmbP in the periplasm of Escherichia coli. Here, we report the highest periplasmic production for this protein so far. Therefore, PelB‐SmbP is an attractive tag for the expression and purification of other target therapeutic proteins in Escherichia coli.

Live-cell protein labeling using a protein tag and a fluorescent probe is a powerful approach for studying protein localization and dynamics. Herein, we engineered a protein tag, PYP-tag, to image ...

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities., Monomeric near-infrared (NIR) fluorescent proteins (FPs) from bacterial phytochromes bring potential advantages, but their brightness in cells is lower than dimeric NIR FPs. Here the authors develop enhanced monomeric NIR FPs enabling imaging across different scales without the trade-off between brightness and monomeric state.

In the past decade, a powerful biotechnological tool for specific in vivo and in vitro labeling of proteins of interest, the SNAP-tag technology, has been proposed as a valid alternative to the classic protein-tags. This self-labeling protein-tag is an engineered variant of the human O6-alkylguanine-DNA-alkyltransferases (hMGMT) enzyme, which specifically reacts with benzyl-guanine (BG) derivatives, maintaining a part of these substrates in the catalytic site, with a one-step reaction. However, given the mesophilic nature of this protein-tag, the general use of this new approach is restricted to mesophilic model systems and mild reaction conditions. To overcome this limitation, it is necessary the exploitation of homologous activities from extremophilic sources, to apply this technology also to (hyper)thermophilic organisms and harsher reaction conditions. To this purpose, it has been first characterized an archaeal alkylguanine-DNA-alkyltransferases (AGT or OGT) from Saccharolobus solfataricus (SsOGT), which presented an exceptional in vitro stability at high temperatures, physical and chemical denaturing agents, making it suitable not only for the in vivo heterologous expression in Escherichia coli, but also in the thermophilic Thermus thermophilus and Sulfolobus islandicus organisms. Furthermore, the growing demand to apply this biotechnological tool to extreme conditions led to look for new protein-tags from hot sources. Recently, new AGTs from Thermotoga neapolitana (TnOGT) and Pyrococcus furiosus (PfuOGT) resulted different from SsOGT in terms of activity and stability, proposing them as potential tool for the in vivo analysis and function of enzymes of interest in these hyperthermophilic model systems.

Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP-tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics., Biochemistry, 60 (33), ISSN:0006-2960, ISSN:1520-4995

To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS.

Protein acetylation is a central event in orchestrating diverse cellular processes. However, current strategies to investigate protein acetylation in cells are often non-specific or lack temporal and magnitude control. Here, we developed an acetylation tagging system, AceTAG, to induce acetylation of targeted proteins. The AceTAG system utilizes bifunctional molecules to direct the lysine acetyltransferase p300/CBP to proteins fused with the small protein tag FKBP12F36V, resulting in their induced acetylation. Using AceTAG, we induced targeted acetylation of a diverse array of proteins in cells, specifically histone H3.3, the NF-κB subunit p65/RelA, and the tumor suppressor p53. We demonstrate that targeted acetylation with the AceTAG system is rapid, selective, reversible, and can be controlled in a dose-dependent fashion. AceTAG represents a useful strategy to modulate protein acetylation and will enable the exploration of targeted acetylation in basic biological and therapeutic contexts.Abstract Figure

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD–RIAD) system. These peptides allow self-assembly based on specific protein–protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD–PTDHRIDD and PAMORIAD/RIAD–PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH–PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO–PTDH complex less PTDH was required for the same performance when compared with the PTDH–PAMO fusion enzyme.

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast d-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. In aqueous two-phase (PEG–phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast d-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast d-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG–phosphate aqueous system.

Sequence-selective chemical modification of DNA by synthetic ligands has been a long-standing challenge in the field of chemistry. Even when the ligand consists of a sequence-specific DNA binding domain and reactive group, sequence-selective reactions by these ligands are often accompanied by off-target reactions. A basic principle to design DNA modifiers that react at specific sites exclusively governed by DNA sequence recognition remains to be established. We have previously reported selective DNA modification by a self-ligating protein tag conjugated with a DNA-binding domain, termed as a modular adaptor, and orthogonal application of modular adaptors by relying on the chemoselectivity of the protein tag. The sequence-specific crosslinking reaction by the modular adaptor is thought to proceed in two steps: the first step involves the formation of a DNA-protein complex, while in the second step, a proximity-driven intermolecular crosslinking occurs. According to this scheme, the specific crosslinking reaction of a modular adaptor would be driven by the DNA recognition process only when the dissociation rate of the DNA complex is much higher than the rate constant for the alkylation reaction. In this study, as a proof of principle, a set of combinations for modular adaptors and their substrates were utilized to evaluate the reactions. Three types of modular adaptors consisting of a single type of self-ligating tag and three types of DNA binding proteins fulfill the kinetic requirements for the reaction of the self-ligating tag with a substrate and the dissociation of the DNA-protein complex. These modular adaptors actually undergo sequence-specific crosslinking reactions exclusively driven by the recognition of a specific DNA sequence. The design principle of sequence-specific modular adaptors based on the kinetic aspects of complex formation and chemical modification is applicable for developing recognition-driven selective modifiers for proteins and other biological macromolecules.

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

Protein tags have been essential for advancing our knowledge of the function of proteins, their localization, and the mapping of their interaction partners. Expressing epitope-tagged proteins has become a standard practice in every life science laboratory and, thus, continues to enable new studies. In recent years, several new tagging moieties have entered the limelight, many of them bringing new functionalities, such as targeted protein degradation, accurate quantification, and proximity labeling. Other novel tags aim at tackling research questions in challenging niches. In this review, we elaborate on recently introduced tags and the opportunities they provide for future research endeavors. In addition, we highlight how the genome-engineering revolution may boost the field of protein tags.

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca2+ indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca2+ fluxes in the primary cilium.

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on localization and function would promote accurate use of tags in experimental setups. Here we examine the effect of Green Fluorescent Protein (GFP) tagging at either the N or C terminus of budding yeast proteins on localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides a step towards broad investigation of protein fusion libraries.

In the last decade, following fluorescent dyes and protein tags, pH sensitive false fluorescent neurotransmitters (FFN) were introduced and were valuable for labeling secretory vesicles and monitoring exocytosis at living cells. In particular, the synthetic analog of neurotransmitters FFN102 was shown to be an electroactive probe. Here, we show that FFN102 is suitable to be used as a bioanalytic probe at the widely used PC12 cell model. FFN102 was uptaken in the secretory vesicles of PC12 cells, partially replacing the endogenous dopamine stored in these vesicles. The different oxidation potentials of dopamine and FFN102 allowed to determine that ca. 12% of dopamine was replaced by FFN102. Moreover, the FFN102 was found to be over released through the initial fusion pore suggesting that it was mostly uptaken in fast diffusion compartment of the vesicles.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

Although many protein fusion tags have been developed for recombinant protein production to improve protein yields or facilitate purification, determining the expression and purification of the fusion protein still remain to be a time-consuming and laborious procedure. In this work, we designed a histidine-rich elastin-like polypeptide (HRELP) fusion tag and found that it could be efficiently expressed in E. coli cells and specifically stained with Pauly's reagent in a couple of minutes post SDS-PAGE analysis. Moreover, in Pauly's reagent-stained polyacrylamide gels, only the bands of HRELP fusion proteins were yellow and could be clearly visualized with little background. Furthermore, both HRELPs and HRELP20-BMP2 fusion protein could be purified by a method of pH shift-mediated inverse transition cycling (ITC). In our opinion, the HRELP established in this study may be considered as a multifunctional protein tag which could make its fusion proteins being quickly detected by Pauly staining and simply purified by pH-triggered ITC in addition to having the potential to sustained release its fusion proteins.

The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation. These approaches range from the direct linkage of N- and C- termini to the connection of amino acid side chains, which can be applied both in reaction vessels and in living systems. In this review, we categorise the cyclisation approaches into chemical methods (e.g. direct backbone cyclisation, native chemical ligation, aldehyde-based ligations, bioorthogonal reactions, disulphide formation), enzymatic methods (e.g. subtiligase variants, sortases, asparaginyl endopeptidases, transglutaminases, non-ribosomal peptide synthetases) and protein tags (e.g. inteins, engineered protein domains for isopeptide bond formation). The features of each approach and the considerations for selecting an appropriate method of cyclisation are discussed., Polypeptide cyclisation can enhance thermal stability, proteolytic resistance and membrane permeability. Cyclisation can be achieved by methods including chemical, enzyme and protein tag approaches. Each has strengths and limitations.

Alkylguanine-DNA-alkyltransferases (AGTs) have a natural role in the protection of DNA from mutations caused by alkylating agents. Their peculiar irreversible self-alkylation reaction led to the development as new tools in the modern biotechnology. SNAP-tag® is a powerful enzyme for the specific labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. This technology has some limitations, as the mesophilic nature of the tag (an engineered variant of the human enzyme) and the needs of purify each substrate. Recently, the SNAP-tag® technology was successfully implemented by the employment of thermostable “SNAP-tag-like” variants from (hyper)thermophilic sources, and by the utilization of BG-substrates containing an azide group to be combined with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. The introduction of these new actors on the scene made possible the expansion of the methodology to in vivo and in vitro harsh reaction conditions, as well as the utilization of more chemical groups in the overall reaction enzyme labelling.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-d-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein–synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.