Search Results (23)

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu²⁺ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His₆) tag in CEA and the unsaturated Cu²⁺ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His₆ tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers. Full article

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal–organic frameworks (MOFs). Zr-based UiO-66-NH₂ MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His₆)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His₆ tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs. Full article

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1–42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer’s disease patients. Aβ(25–35) is the shortest peptide that retains the toxicity of Aβ(1–42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1–42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu²⁺, Fe³⁺ and Zn²⁺. In this work, we show that the fluorescence of Aβ(1–42) HiLyte^TM-Fluor555 can be used to monitor hexa-histidine peptide (His₆) interaction with Aβ(1–42). The formation of His₆/Aβ(1–42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His₆ block the increase in the fluorescence of Aβ(1–42) HiLyte^TM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His₆-tag provides a high-affinity site for the binding of Aβ(1–42) and Aβ(25–35) peptides to the human recombinant cytochrome b₅ reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His₆-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets. Full article

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar K_d) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications. Full article

Molecular immobilization and recognition are two key events for the development of biosensors. The general ways for the immobilization and recognition of biomolecules include covalent coupling reactions and non-covalent interactions of antigen–antibody, aptamer–target, glycan–lectin, avidin–biotin and boronic acid–diol. Tetradentate nitrilotriacetic acid (NTA) is one of the most common commercial ligands for chelating metal ions. The NTA–metal complexes show high and specific affinity toward hexahistidine tags. Such metal complexes have been widely utilized in protein separation and immobilization for diagnostic applications since most of commercialized proteins have been integrated with hexahistidine tags by synthetic or recombinant techniques. This review focused on the development of biosensors with NTA–metal complexes as the binding units, mainly including surface plasmon resonance, electrochemistry, fluorescence, colorimetry, surface-enhanced Raman scattering spectroscopy, chemiluminescence and so on. Full article

Genes encoding endolysins were identified and cloned from three different Escherichia coli bacteriophages, 10-24(13), PBEC30, and PBEC56. Putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with amphipathic natures were predicted from the three endolysins. Each gene was cloned and expressed as hexahistidine-tagged forms, and the products were purified and characterized. The purified endolysins exhibited antibacterial activities against a variety of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Their antibacterial activities were improved by N-terminal fusion with an antimicrobial peptide, cecropin A. Minimum inhibitory concentrations (MIC) were as low as 4 μg/mL, depending on the targeted strain. The endolysins’ enzymatic activities were not affected by changes in pH at ranges from 5 to 10 and were stable at temperatures between 4 and 65 °C. The in vivo efficacies of the three endolysins were also demonstrated using Galleria melonella for infection models. Full article

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His₆-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His₆-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His₆-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His₆-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme. Full article

The chemical synthesis of heterocycles typically requires elevated temperature and acid or base addition to form the desired product. Moreover, these reactions often involve hazardous reagents, which is why biocatalytic routes for heterocycle formation have gained increasing attention. In recent years, several enzymes belonging to the amidohydrolase superfamily have been identified to generate heterocycles via cyclocondensation reactions. Of particular interest is the amidohydrolase MxcM, which catalyzes the formation of an imidazoline moiety in the biosynthesis of the anti-inflammatory natural product pseudochelin A. In this study, we present a concept for the immobilization of this enzyme using a fused hexahistidine tag for fixation onto a solid, porous carrier. Notably, the immobilization improves the enzyme’s tolerance to organic solvents. The immobilized MxcM exhibits a residual activity of 169% in the polar solvent acetonitrile compared to the free enzyme, and the storage stability in the presence of 20 vol% acetonitrile was ameliorated. In addition, an immobilized enzyme reactor (IMER) was designed that can be operated under flow conditions. The MxcM-IMER retains its biocatalytic activity and mechanic stability over the tested operation time. These results provide important insights for the integration of heterocycle-forming amidohydrolases in chemical processes. Full article

The self-assembling of nanosized materials is a promising field for research and development. Multiple approaches are applied to obtain inorganic, organic and composite nanomaterials with different functionality. In the present work, self-assembling nanocomplexes (NCs) were prepared on the basis of enzymes and polypeptides followed by the investigation of the influence of low-molecular weight biologically active compounds on the properties of the NCs. For that, the initially possible formation of catalytically active self-assembling NCs of four hydrolytic enzymes with nine effectors was screened via molecular modeling. It allowed the selection of two enzymes (hexahistidine-tagged organophosphorus hydrolase and penicillin acylase) and two compounds (emodin and naringenin) having biological activity. Further, such NCs based on surface-modified enzymes were characterized by a batch of physical and biochemical methods. At least three NCs containing emodin and enzyme (His₆-OPH and/or penicillin acylase) have been shown to significantly improve the antibacterial activity of colistin and, to a lesser extent, polymyxin B towards both Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli). Full article

Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties. Full article

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery. Full article

Mycotoxins are dangerous compounds and find multiple routes to enter living bodies of humans and animals. To solve the issue and degrade the toxicants, (bio)catalytic processes look very promising. Hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) is a well-studied catalyst for degradation of organophosphorus neurotoxins and lactone-containing quorum-sensing signal molecules. Moreover, the catalytic characteristics in hydrolysis of several mycotoxins (patulin, deoxynivalenol, zearalenone, and sterigmatocystin) were studied in this investigation. The best Michaelis constant and catalytic constant were estimated in the case of sterigmatocystin and patulin, respectively. A possible combination of His₆-OPH with inorganic sorbents treated by low-temperature plasma was investigated. Further, enzyme–polyelectrolyte complexes of poly(glutamic acid) with His₆-OPH and another enzymatic mycotoxin degrader (thermolysin) were successfully used to modify fiber materials. These catalytically active prototypes of protective materials appear to be useful for preventing surface contact and exposure to mycotoxins and other chemicals that are substrates for the enzymes used. Full article

To obtain composite fiber materials with antibacterial properties, the samples of bacterial cellulose (BC), polylactide (PLA) and usual fibers (FM) were modified by poly-ε-caprolactone or polyhydroxybutyrate and then functionalized by the enzyme-polyelectrolyte complex of quorum-quenching enzymes, such as hexahistidine-tagged organophosphorus hydrolase with poly(glutamic acid) or by suspension of tantalum nanoparticles (Ta NPs) in ethanol. The structures of the composite fibers were analyzed using scanning electron microscopy. It was shown that the introduction of additional natural polymers into the matrix of BC, PLA and FM resulted in decreasing of the structural porosity. Comparative studies of the antibacterial activity of the composite materials were carried out using Escherichia coli and Bacillus subtilis cells. The decrease of adenosine triphosphate concentration in cell samples loaded onto fiber materials was applied as a measurable characteristic of antibacterial effect typical for the new fiber materials. The profound improvement of antibacterial activity was determined in composite materials with polyhydroxybutyrate and Ta NPs. Full article

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids. Full article

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N-acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His₆-OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against Escherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His₆-OPH complexes as antimicrobial protectants. Full article