Metal Chelate Affinity Chromatography Research Articles

Новый рекомбинантный ингибитор РНКаз LoRI для применения in vitro

The novel ribonuclease inhibitor LoRI is a 63 kDa recombinant protein optimized for high-throughput expression in E. coli and purification by metal chelate affinity chromatography (IMAC). The product was obtained by N-terminal fusion of mouse placental RNase inhibitor polypeptide to a thioredoxin module. Advantage of the engineering strategy in terms of protein structure and function was predicted in silico. Under laboratory settings, the yield of purified soluble recombinant product was about 12 mg per 1 L of expression bacterial culture. By RNase inhibition capacity in vitro, the product is comparable or superior to a commercial reference. The kinetic data comply with Lineweaver-Burk model.

CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF ATRPS6B1 RECOMBINANT PROTEIN

Living organisms must adapt their growth and development in response to external factors like stress and nutrient availability throughout their lifespan. Consequently, they have evolved various regulatory pathways to enhance environmental perception and facilitate necessary metabolic adjustments. These pathways involve key proteins that, under stress and nutrient limitation, initiate anabolic and catabolic cellular processes. One critical pathway present in all eukaryotes involves the protein kinase TOR (Target of rapamycin), a large kinase that governs numerous biological processes, including the activation of S6K kinase, which phosphorylates the S6 protein (RPS6). Crystal structures have revealed the precise location of RPS6 within the 40S ribosomal subunit, highlighting structural features such as the C-terminal helix containing multiple phosphorylation sites. RPS6 primarily regulates mechanisms controlling cell growth and division. In plants, eS6 is encoded by two conserved genes, and its activation and phosphorylation are closely linked to S6Ks. Recent studies emphasize the significance of S6 protein phosphorylation as a key event in signaling pathways related to cell growth and survival factors, crucially regulating translation initiation and protein synthesis. S6 protein can be phosphorylated on various serine and threonine residues by S6K kinase, and further research shows its interaction with other molecules and proteins, expanding its functional roles in cell signaling. Further exploration of ribosomal protein S6 could unveil new insights into its molecular interactions, roles in cellular physiology and pathophysiology, and potential applications in enhancing plant biomass and yield. This study involved cloning and site-directed mutagenesis of cDNA for the second isoform of AtRPS6 protein (AtRPS6B), followed by expression of natural and phosphomimetic forms of the protein in E. coli ArcticExpress (DE3) cells. The proteins were then purified using metal-chelate affinity chromatography (IMAC), and their presence and degree of purification were confirmed via Western blotting. These findings contribute to the broader research context surrounding ribosomal protein S6. RPS6, a key effector in the TOR signaling pathway, plays a crucial role in regulating ribosome biosynthesis by controlling transcription and translation processes. Phosphorylation of RPS6, indicative of S6K activity, is associated with actively dividing cells. However, the precise role of RPS6 in ribosome biosynthesis regulation remains a subject of ongoing investigation. The data obtained from the study on AtRPS6B expand our understanding of the molecular mechanisms underlying ribosomal biosynthesis regulation. These insights may significantly contribute to our knowledge of the interplay between signaling pathways, ribosomal proteins, cellular metabolism, and offer new perspectives for therapeutic interventions targeting cellular processes associated with ribosomes.

Reconstitution of the in vitro STUB1-mediated ubiquitination reaction system for α-1 antitrypsin mutant Z protein

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation

BackgroundTraditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials.ResultsWe demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v).ConclusionLysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Recombinant production of antibody antigen-binding fragments with an N-terminal human growth hormone tag in mammalian cells

Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at ∼4 μg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.

Purification and biochemical characterization of catalase that confers protection against hydrogen peroxide induced by stressful desert environment: the Camelus Dromedarius kidney catalase

Camel is continually exposed to stressful desert environment that enhances generation of reactive oxygen species, including hydrogen peroxide (H2O2). Catalase plays an important role in detoxification of H2O2. A highly active catalase from camel kidney was purified to homogeneity, with a specific activity of 1,774,392 U/mg protein, using ion exchange and metal chelate affinity chromatography. The molecular weight of the enzyme was 268 kDa consisting of four identical subunits of 63 kDa. The enzyme showed higher optimum temperature (45 °C) and higher activation energy (4.37 kJ mol−1). The thermodynamic parameters, ΔH, ΔG and ΔS, were determined. The effect of various metal ions and chemicals on enzyme activity was investigated. Km , Vmax , kcat and kcat /Km values for H2O2 were found to be 46 mM, 10,715,045 U/mg, 48,265,968 s−1 and 2,966,562 s−1 mM−1, respectively. Camel kidney catalase displayed higher affinity efficiency for H2O2 and can protect reduced glutathione (GSH) from oxidation by H2O2. Sodium azide was found to be a noncompetitive inhibitor of enzyme with Ki and IC50 of 17.88 µM and 20.94 µM, respectively. Camel catalase showed unique biochemical properties. Interestingly, camel catalase can protect molecules (GSH) and organ functions (kidney) from the toxic effects of H2O2 induced by stressful desert environment.

ISOLATION AND STRUCTURE ANALYSIS OF THE MUTANT FORM OF N-TERMINAL CATALYTIC MODULE OF BOS TAURUS TYROSYL-tRNA SYNTHETASE WITH REPLACEMENT OF Trp 40 AND Trp 87 BY ALANINE

Aim. Isolation and analysis of the structure of the mutant monotryptophan protein mini BtTyrRS for study of conformational changes of the enzyme at the stage of interaction with tRNA using fluorescence spectroscopy and determination of the effect of tryptophan residues in position 40 and 87 in its structure on the functional properties of the enzyme. Methods. Electrophoresis, metal-chelating affinity chromatography, fluorescence spectroscopy, spatial structure modeling. Results. It was found that the replacement of two codons of Trp by codons of Ala in the cloned cDNA mini BtTyrRS does not affect the synthesis of the mutant form of the enzyme in E. coli strain BL21 (DE3) pLysE. The yield of affinity purified protein on Ni-NTA agarose is on average 3.5 mg per 100 ml of culture medium. Computer modeling of the structure and fluorescence spectroscopy of the monotryptophan form of mini BtTyrRS indicates a compact structure of the mutant enzyme, in which Trp 283 is in an immobilized microenvironment. Conclusions. Affinity purified on Ni-NTA agarose mutant monotryptophan protein mini TyrRS have been obtained which is suitable for fluorescent studies of structural-dynamic and functional properties of the enzyme.

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Studying the immune response genes expression regulation of Drosophila melanogaster

The main control of the synthesis of the antimicrobial peptides in the cells of Drosophila melanogaster is carried out by two signalling pathways IMD and Toll, which include the classic transcription factors of the NF-kB family (Dif, Dorsal and Relish). The Toll pathway is activated in response to gram-positive bacteria and fungi, and the IMD pathway is activated in response to gram-negative bacteria. In addition to transcription factors, the transcriptional apparatus of the cells includes the co-activators of transcription, such as the SAYP protein, which was described earlier in our laboratory. Co-activators do not bind directly to DNA, but assemble different parts of the transcriptional complex, which includes chromatin-remodelling complex, the specific transcription factors and the general transcription factors, which bind to RNA Pol II. SAYP homologues were found in the genomes of all multicellular animals. The protein mediates various functions of ontogenesis. We found that knockdown of SAYP leads to a decrease in expression of the specific genes of the IMD and Toll immune response pathways. In addition, the proteomic analysis of proteins associated with SAYP revealed sequences of another transcription factor, which is called DEAF-1. This protein is also involved in the immune response and ontogenesis. These prerequisites determined the task for studying the interactions of SAYP, Dif, Dorsal, Relish and DEAF-1 factors and their influence on the genes expression activation. We have obtained antibodies to the described factors. The most optimal epitopes (presumably exposed and accessible for recognition) were selected; the corresponding fragments of the cDNA genes were inserted into the expression vector and the conditions of expression in E.coli cells were matched. Recombinant proteins were produced and purified by metal-chelate affinity chromatography using Ni-Sepharose. The presence of these proteins in the fractions was confirmed by MALDI. The proteins have been used to immunize rabbits. The antibodies have been affinity purified from sera. The specificity of the antibodies was verified by Western blot analysis of the Drosophila embryonic nuclear extract. We have developed a model system for activating the IMD and Toll pathways. Escherichia coli and Micrococcus luteus cells were grown, washed and resuspended in water. The suspensions were inactivated and added to the S2 D. melanogaster cell cultures. Using qPCR, we have observed the activation of the immune response genes. Using the model system, we checked the recruitment of two model genes (Cecropin A1, Metchnikowin) to the promoters of the studied transcription co-activator. Chromatin immunoprecipitation has shown the binding of studied co-factor to gene promoters upon their activation. Immunoprecipitation using the obtained antibodies to DEAF-1 and SAYP has shown the co-precipitation of DEAF-1 protein with SAYP. This indicates that these proteins may jointly participate in the regulation of the immune response genes expression. We are currently looking for the individual domains of these proteins that mediate their interactions.

Design, Dimerization, and Recombinant Production of MCh-AMP1-Derived Peptide in Escherichia coli and Evaluation of Its Antifungal Activity and Cytotoxicity.

Fungal species resistant to current antifungal agents are considered as a serious threat to human health, the dilemma that has dragged attentions toward other sources of antifungals such as antimicrobial peptides (AMPs). In order to improve biological activity of a recently described antifungal peptide MCh-AMP1 from Matricaria chamomilla flowers, MCh-AMP1dimer (DiMCh-AMP1), containing 61 amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed and expressed in Escherichia coli, and its structure was analyzed using bioinformatics tools. DiMCh-AMP1 synthetic gene was cloned into pET-28a expression vector, which was then used to transform E. coli BL21 (DE3) strain. His-tag purification was achieved using metal-chelate affinity chromatography. Because there is no methionine residue in the DiMCh-AMP1 sequence, cyanogen bromide was successfully used to separate the target product from the tag. Reverse-phase high-performance liquid chromatography was used as the final step of purification. Results showed that recombinant peptide was produced in considerable amounts (0.9 mg/L) with improved antifungal activity toward both yeasts and molds compared to its monomeric counterpart. The minimum inhibition concentration and minimum fungicidal concentration values of DiMCh-AMP1 against Candida and Aspergillus species were reported in the range of 1.67-6.66 μM and 3.33-26.64 μM, respectively. Our results showed that while antifungal activity of dimerized peptide was improved considerably, its cytotoxicity was decreased, implying that DiMCh-AMP1 could be a potential candidate to design an effective antifungal agent against pathogenic yeasts and molds.

Expression and physicochemical characterization of an N-terminal polyhistidine-tagged phosphotriesterase from the soil bacterium Brevundimonas diminuta

Abstract In this study, a truncated organophosphate-degrading (opd) gene encoding a Brevundimonas diminuta phosphotriesterase (BdPTE) without its leader peptide was artificially synthesized and cloned into the expression vector pQE-30 to yield pQE-BdPTE. The His6-tagged 37.8-kDa enzyme was purified from the cell-free extract of isopropyl thio-β- d -galactoside (IPTG)-induced E. coli M15 (pRep4) cells harboring pQE-BdPTE to near homogeneity by metal chelate affinity chromatography. The assay conditions for His6-tagged BdPTE activity were optimized at 60 °C, pH 8.0, and 1.0 mM Co2+ ion. Kinetic analysis of His6-tagged BdPTE-catalyzed hydrolysis of diethyl p-nitrophenyl phosphate (paraoxon-ethyl) showed that its Km and kcat values were 21.9 ± 1.4 μM and 311.6 ± 12.3 s−1, respectively. Also, the recombinant enzyme had the ability to efficiently degrade the two most commonly used OP pesticides, ethion and fenthion, in Taiwan. His6-tagged BdPTE with 1 mM Co2+ was very stable during the storage period of 35 days at 4 °C. Urea- and guanidine hydrochloride (GdnHCl)-induced denaturation studies showed that the purified enzyme had [denaturant]0.5,N–U values of 6.7 and 3.4 M, respectively. Notably, His6-tagged BdPTE did exhibit some degree of tolerance to a variety of water-miscible organic co-solvents. Dimethyl sulfoxide (DMSO), acetone, dimethylformamide (DMF), and ethanol were obviously harmful to the hydrolytic activity of the enzyme at concentrations above 50% (v/v), while acetonitrile was the highest tolerated co-solvent. The organic solvent-tolerant feature verifies His6-tagged BdPTE highly suitable as a remediation agent for the biodegradation of non-polar OP pesticides.

Structural characterization of calcium-binding sunflower seed and peanut peptides and enhanced calcium transport by calcium complexes in Caco-2 cells.

Peptide-Ca complexes can promote Ca absorption. The present study aimed to determine the transport mechanism and structural characteristics of sunflower seed and peanut peptides with high Ca binding capacity with respect to developing third-generation Ca supplements and functional food ingredients. High Ca-binding fractions of 1-3 kDa sunflower seed peptide (SSP4 ) and ≥ 10 kDa peanut peptide (PP1 ) had higher amount of Ca transported than CaCl2 and two hydrolyzed proteins in Caco-2 cells. SSP4 and PP1 were separated by Ca ion metal chelate affinity chromatography, and high Ca-binding fractions were observed for SSP4 -P2 and PP1 -P2 . The amino acid sequences of SSP4 -P2 and PP1 -P2 were characterized by high-performance liquid chromatography-electrospray ionization-time of flight mass spectrometry. Seven and eight peptides were identified from SSP4 -P2 and PP1 -P2 , respectively. These peptides had molecular weights ranging from 1500 Da to 2500 Da and a large number of characteristic amino acid sequences, such as EEEQQQ, EQ-QQQ-QQ, QQ-QQQQQ, E-EEE, EE-EEQ, RR, Q-QQ-QQQ, EE-EQ-EE-Q, QQ-QQQQ, and Q-QQQQ, where 'E' is glutamic acid and 'Q' is glutamine. SSP4 and PP1 can promote Ca transport in Caco-2 cells without affecting cell permeability. The amino acid sequences of SSP4 -P2 and PP1 -P2 with high Ca-binding abilities contain characteristic sequences, such as continuous glutamic acid and glutamine, and have low molecular weights. © 2020 Society of Chemical Industry.

Influence of His6 Sequence on the Properties of Formate Dehydrogenase from Bacterium Pseudomonas sp. 101

NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cofactor regeneration. The efficient use of FDH in such systems requires simple and fast enzyme purification. Metal-chelate affinity chromatography is widely used for such purposes. The method requires the presence of at least six His residues at N- or C-terminus of protein. The addition of extra His residues can affect enzyme properties. The computer modeling of the structure of FDH from bacterium Pseudomonas sp. 101 with different positions of His6 sequence showed that the optimal case is His-tag at N-terminus. Three types of PseFDH with His6 were prepared: wild-type NAD+-dependent enzyme and two mutant NADP+-specific forms. New PseFDHs were obtained as homogeneous preparations through a one-step purification procedure. The comparison of PseFDHs with and without His-tag showed that they have similar kinetic properties.

Corrigendum: Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

[This corrects the article DOI: 10.3389/fcimb.2018.00237.].

Heterologous overexpression of active hexokinases from microsporidia Nosema bombycis and Nosema ceranae confirms their ability to phosphorylate host glucose.

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01mM and kcat 1726min-1 for glucose, and Km 0.39 ± 0.05mM and kcat 1976min-1 for ATP, at pH8.8. N. ceranae HK showed Km 0.3 ± 0.04mM and kcat 3293min-1 for glucose, and Km 1.15 ± 0.11mM and kcat 3732min-1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

Chitosan Co-polymeric nanostructures for catalase immobilization

Catalase is an industrial enzyme used in different areas including medical, textile, food and pharmaceutic industries. In the study, synthetic-natural copolymer in nanosize was synthesized using 2-hydroxyethyl methacrylate (HEMA) as a synthetic material and chitosan (CTS) as a natural material. Immobilized metal-chelate affinity chromatography (IMAC) material was prepared by chelating Cu(II) ions onto p(HEMA-CTS) nanostructures. Characterization studies such as Zeta sizer analysis, Fourier transform infra-red (FTIR) spectroscopy, scanning electron microscopy (SEM), and thermal gravimetric analysis (TGA) were performed during the preparation of IMAC nanostructures. The effects of some parameters (time, polymer amount, pH, initial catalase concentration, ionic strength and temperature) were identified and optimum immobilization conditions were determined as 75 min, 0.1 mg p(HEMA-CTS)-Cu, pH 6.0, and 25 °C. Maximum amount of immobilized catalase was detected as 11.29 g/g p(HEMA-CTS)-Cu. Immobilized catalase was desorbed successfully and it was detected that p(HEMA-CTS)-Cu nanostructures can be used repeatedly in catalase immobilization. Immobilization changed optimum temperature value from 37 °C to 25 °C while it did not change the optimum pH for catalase activity. Furthermore, it was detected that kinetic parameters of catalase changed by immobilization. Finally, operational and storage stabilities of immobilized catalase were investigated.

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4–8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.

Detection of Brucella abortus by immunofluorescence assay using anti outer membrane protein of 19 kDa antibody.

Brucellosis in humans is one of the most prevalent zoonotic diseases around the world with more than 500,000 new cases per year. It is a weakening disease that requires long-term antibiotic treatment, often resulting in permanent and disabling consequences. Outer membrane proteins (OMPs) of Brucella, which are non-lipopolysaccharide (LPS) antigens, have been used for the diagnostic kits of brucellosis and vaccine design. The aim of this study was to identify Brucella abortus with an immunofluorescence (IF) test using an antibody against recombinant outer membrane protein (OMP) of 19 kDa of this bacterium. The OMP19 gene of Brucella spp. was synthesized, cloned and expressed in Escherichia coli cells. The OMP19 protein was purified by metal chelate affinity chromatography and subsequently used for the immunization of rabbits to produce a polyclonal antibody. Then, this antibody was conjugated to fluorescein isothiocyanate (FITC) and used for the detection of Brucella by an IF test. Also, the sensitivity and specificity of this antibody for the diagnosis of clinical isolates was calculated. Outer membrane protein 19 was expressed well and reacted with a commercial antiserum against His-tag in an immunoblot assay. Polyclonal antibodies obtained from the serum of rabbits immunized with the purified protein showed strong reactivity in the enzyme-linked immunosorbent assay (ELISA). Moreover, the polyclonal antibody conjugated to FITC was able to properly identify Brucella abortus. Sensitivity and specificity of this IF test in comparison with a polymerase chain reaction (PCR) assay was 84.2% and 50%, respectively. This high-titer antibody could potentially be valuable for the specific diagnostic test of brucellosis.