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Analytical Methods : Advancing Methods... Jun 2024The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10...
The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10 kDa cell filtrate protein (CFP-10) and 6 kDa early secretory antigenic target (ESAT-6) proteins from the complex (MTC) in broth culture media with the use of the 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Conventional matrices, α-cyano-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), were also used for comparison. After nanodiamond (ND) extraction, the sequence coverage of the CFP-10 protein was 87% when CHCA and DHB matrices were used, and the ESAT-6 protein was not detected. On the other hand, the sequence coverage for ND-extracted CFP-10 and ESAT-6 could reach 94% and 100%, respectively, when the Cl-CCA matrix was used and with the removal of interference from bovine serum albumin (BSA) protein and α-crystallin (ACR) protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also adopted to analyze the protein mass spectra. A total of 6 prominent ion signals were observed, including ESAT-6 protein peaks at mass-to-charge ratios (/) of ∼7931, ∼7974, ∼9768, and ∼9813 and CFP-10 protein peaks at / of ∼10 100 and ∼10 660. The ESAT-6 ion signals were always detected concurrently with CFP-10 ion signals, but CFP-10 ion signals could be detected alone without the ESAT-6 ion signals. Furthermore, the newly found ESAT-6 peaks were also confirmed using a Mag-Beads-Protein G kit with an ESAT-6 antibody to capture the ESAT-6 protein, which was also consistent with the sequence coverage analysis.
Topics: Mycobacterium tuberculosis; Bacterial Proteins; Nanodiamonds; Antigens, Bacterial; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 38804556
DOI: 10.1039/d4ay00314d -
Archives of Microbiology Jun 2024Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various...
Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.
Topics: Humans; Animals; Female; Cloning, Molecular; Anti-Inflammatory Agents; Mice; Recombinant Proteins; MCF-7 Cells; Breast Neoplasms; Escherichia coli; Serratia; Metalloproteases; Antineoplastic Agents; Bacterial Proteins
PubMed: 38907853
DOI: 10.1007/s00203-024-04051-y -
Science (New York, N.Y.) Nov 2023These and other ancient plants may provide alternatives to chemical insecticides.
These and other ancient plants may provide alternatives to chemical insecticides.
Topics: Ferns; Insecticides; Crops, Agricultural; Plant Proteins; Plants, Genetically Modified; Zea mays; Pest Control, Biological; Glycine max
PubMed: 37995240
DOI: 10.1126/science.adn0185 -
Polish Journal of Microbiology Jun 2024Proteases derived from demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study...
Proteases derived from demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na, K, Mg, and Cumetal ions. The kinetic parameters were determined with a value of 3.13 mg/ml and a value of 3.28 × 10 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from sp. CNXK100.
Topics: Streptomyces; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Temperature; Bacterial Proteins; Peptide Hydrolases; Molecular Weight; Metals
PubMed: 38678439
DOI: 10.33073/pjm-2024-014 -
Food Chemistry Aug 2024Edible insects represent a great alternative protein source but food neophobia remains the main barrier to consumption. However, the incorporation of insects as...
Influence of the processing on composition, protein structure and techno-functional properties of mealworm protein concentrates produced by isoelectric precipitation and ultrafiltration/diafiltration.
Edible insects represent a great alternative protein source but food neophobia remains the main barrier to consumption. However, the incorporation of insects as protein-rich ingredients, such as protein concentrates, could increase acceptance. In this study, two methods, isoelectric precipitation and ultrafiltration-diafiltration, were applied to produce mealworm protein concentrates, which were compared in terms of composition, protein structure and techno-functional properties. The results showed that the protein content of the isoelectric precipitation concentrate was higher than ultrafiltration-diafiltration (80 versus 72%) but ash (1.91 versus 3.82%) and soluble sugar (1.43 versus 8.22%) contents were lower. Moreover, the protein structure was affected by the processing method, where the ultrafiltration-diafiltration concentrate exhibited a higher surface hydrophobicity (493.5 versus 106.78 a.u) and a lower denaturation temperature (161.32 versus 181.44 °C). Finally, the ultrafiltration-diafiltration concentrate exhibited higher solubility (87 versus 41%) and emulsifying properties at pH 7 compared to the concentrate obtained by isoelectric precipitation.
Topics: Ultrafiltration; Animals; Hydrophobic and Hydrophilic Interactions; Insect Proteins; Tenebrio; Chemical Precipitation; Solubility; Hydrogen-Ion Concentration; Food Handling
PubMed: 38581785
DOI: 10.1016/j.foodchem.2024.139177 -
Journal of Global Antimicrobial... Jun 2024
Topics: beta-Lactamases; Bacterial Proteins; Anti-Bacterial Agents; Microbial Sensitivity Tests; Humans; Chloramphenicol; Enterobacteriaceae; Carbapenem-Resistant Enterobacteriaceae; Enterobacteriaceae Infections
PubMed: 38417738
DOI: 10.1016/j.jgar.2024.02.003 -
Protein Expression and Purification Sep 2024Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant...
Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.
Topics: Carbon-Nitrogen Ligases; Staphylococcus aureus; Bacterial Proteins; Gene Expression; Molecular Docking Simulation; Recombinant Proteins
PubMed: 38833752
DOI: 10.1016/j.pep.2024.106520 -
Biochimie May 2024Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals,...
Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
Topics: Azoles; Fungal Proteins; Drug Resistance, Fungal; Antifungal Agents; Candida albicans; Membrane Transport Proteins; Candida glabrata; ATP-Binding Cassette Transporters
PubMed: 38158037
DOI: 10.1016/j.biochi.2023.12.007 -
Enzyme and Microbial Technology Aug 2024The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis...
The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.
Topics: Enzymes, Immobilized; Bacterial Proteins; Enzyme Stability; alpha-L-Fucosidase; Recombinant Fusion Proteins; Glycoside Hydrolases
PubMed: 38581868
DOI: 10.1016/j.enzmictec.2024.110445 -
Biomolecules Mar 2024The identification of the hormone erythropoietin (EPO), which regulates red blood cell production, and its development into a pharmaceutical-grade product to treat... (Review)
Review
The identification of the hormone erythropoietin (EPO), which regulates red blood cell production, and its development into a pharmaceutical-grade product to treat anemia has been not only a herculean task but it has also been the first of its kind. As with all the successes, it had "winners" and "losers", but its history is mostly told by the winners who, over the years, have published excellent scientific and divulgate summaries on the subject, some of which are cited in this review. In addition, "success" is also due to the superb and dedicated work of numerous "crew" members, who often are under-represented and under-recognized when the story is told and often have several "dark sides" that are not told in the polished context of most reviews, but which raised the need for the development of the current legislation on biotherapeutics. Although I was marginally involved in the clinical development of erythropoietin, I have known on a personal basis most, if not all, the protagonists of the saga and had multiple opportunities to talk with them on the drive that supported their activities. Here, I will summarize the major steps in the development of erythropoietin as the first bioproduct to enter the clinic. Some of the "dark sides" will also be mentioned to emphasize what a beautiful achievement of humankind this process has been and how the various unforeseen challenges that emerged were progressively addressed in the interest of science and of the patient's wellbeing.
Topics: Animals; Humans; Anemia; Erythropoietin; History, 20th Century; History, 21st Century
PubMed: 38672425
DOI: 10.3390/biom14040408