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https://www.readbyqxmd.com/read/28330113/protein-fusion-tags-for-efficient-expression-and-purification-of-recombinant-proteins-in-the-periplasmic-space-of-e-coli
#1
REVIEW
Ajamaluddin Malik
Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical...
June 2016: 3 Biotech
https://www.readbyqxmd.com/read/28323277/an-alternative-polysaccharide-uptake-mechanism-of-marine-bacteria
#2
Greta Reintjes, Carol Arnosti, Bernhard M Fuchs, Rudolf Amann
Heterotrophic microbial communities process much of the carbon fixed by phytoplankton in the ocean, thus having a critical role in the global carbon cycle. A major fraction of the phytoplankton-derived substrates are high-molecular-weight (HMW) polysaccharides. For bacterial uptake, these substrates must initially be hydrolysed to smaller sizes by extracellular enzymes. We investigated polysaccharide hydrolysis by microbial communities during a transect of the Atlantic Ocean, and serendipitously discovered-using super-resolution structured illumination microscopy-that up to 26% of total cells showed uptake of fluorescently labelled polysaccharides (FLA-PS)...
March 21, 2017: ISME Journal
https://www.readbyqxmd.com/read/28320881/lpto-pg0027-is-required-for-lipid-a-1-phosphatase-activity-in-porphyromonas-gingivalis-w50
#3
Minnie Rangarajan, Joseph Aduse-Opoku, Ahmed Hashim, Graham McPhail, Zofia Luklinska, M Florencia Haurat, Mario F Feldman, Michael A Curtis
Porphyromonas gingivalis produces outer-membrane vesicles (OMVs) rich in virulence factors including cysteine-proteases and A-LPS, one of the two lipopolysaccharides (LPS) produced by this organism. Previous studies had suggested that A-LPS together with PG0027, an outer-membrane (OM) protein, may be involved in OMV-formation. Their roles in this process were examined using parent W50 and mutant ΔPG0027 strains. Inactivation of PG0027 caused a reduction in yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and ΔPG0027 strains were analysed by matrix-assisted laser-desorption-ionisation-time of flight mass spectrometry (MALDI-TOF- MS)...
March 20, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28320377/application-of-an-e-coli-signal-sequence-as-a-versatile-inclusion-body-tag
#4
Wouter S P Jong, David Vikström, Diane Houben, H Bart van den Berg van Saparoea, Jan-Willem de Gier, Joen Luirink
BACKGROUND: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form...
March 21, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28318016/the-transporter-synpam71-is-located-in-the-plasma-membrane-and-thylakoids-and-mediates-manganese-tolerance-in-synechocystis-pcc6803
#5
Chiara Gandini, Sidsel Birkelund Schmidt, Søren Husted, Anja Schneider, Dario Leister
Manganese (Mn) is an essential constituent of photosystem II (PSII) and therefore indispensable for oxygenic photosynthesis. Very little is known about how Mn is transported, delivered and retained in photosynthetic cells. Recently, the thylakoid-localized transporter PAM71 has been linked to chloroplast Mn homeostasis in Arabidopsis thaliana. Here, we characterize the function of its homolog in Synechocystis (SynPAM71). We used a loss-of-function line (ΔSynPAM71), wild-type (WT) cells exposed to Mn stress and strains expressing a tagged variant of SynPAM71 to characterize the role of SynPAM71 in cyanobacterial Mn homeostasis...
March 20, 2017: New Phytologist
https://www.readbyqxmd.com/read/28316480/inducible-spy-transcription-acts-as-a-sensor-for-envelope-stress-of-salmonella-typhimurium
#6
Seon Mi Jeong, Hwa Jeong Lee, Yoon Mee Park, Jin Seok Kim, Sang Dae Lee, Iel Soo Bang
Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (spheroplast protein y)...
2017: Korean Journal for Food Science of Animal Resources
https://www.readbyqxmd.com/read/28302513/expression-purification-and-enzymatic-characterization-of-undecaprenyl-pyrophosphate-phosphatase-from-vibrio-vulnificus
#7
Hsin-Yang Chang, Chia-Cheng Chou, Mao-Lun Wu, Andrew H J Wang
Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E...
March 14, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28300394/an-asymmetric-conformational-change-in-lacy
#8
Irina Smirnova, Vladimir Kasho, Xiaoxu Jiang, H Ronald Kaback
Galactoside/H(+) symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding...
March 23, 2017: Biochemistry
https://www.readbyqxmd.com/read/28300142/late-assembly-of-the-vibrio-cholerae-cell-division-machinery-postpones-septation-to-the-last-10-of-the-cell-cycle
#9
Elisa Galli, Evelyne Paly, François-Xavier Barre
Bacterial cell division is a highly regulated process, which involves the formation of a complex apparatus, the divisome, by over a dozen proteins. In the few model bacteria in which the division process was detailed, divisome assembly occurs in two distinct steps: a few proteins, including the FtsZ tubulin-like protein, form a membrane associated contractile ring, the Z-ring, at ~30% of the cell cycle. The Z-ring serves as a scaffold for the recruitment of a second series of proteins, including integral membrane and periplasmic cell wall remodelling enzymes, at ~50% of the cell cycle...
March 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28295618/mxaj-structure-reveals-a-periplasmic-binding-protein-like-architecture-with-unique-secondary-structural-elements
#10
Jin Myung Choi, Thinh-Phat Cao, Si Wouk Kim, Kun Ho Lee, Sung Haeng Lee
MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome cL . Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α-helices and six β-strands, and resembles the "bi-lobate" folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a β-strand around the hinge region supporting the ligand-binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules...
March 13, 2017: Proteins
https://www.readbyqxmd.com/read/28295106/structure-electrocatalysis-and-dynamics-of-immobilized-cytochrome-pcch-and-its-microperoxidase
#11
Célia M Silveira, María A Castro, Joana M Dantas, Carlos Salgueiro, Daniel H Murgida, Smilja Todorovic
Geobacter sulfurreducens cells have the ability to exchange electrons with conductive materials, and the periplasmic cytochrome PccH plays an essential role in the direct electrode-to-cell electron transfer in this bacterium. It has atypically low redox potential and unique structural features that differ from those observed in other c-type cytochromes. We report surface enhanced resonance Raman spectroscopic and electrochemical characterization of the immobilized PccH, together with molecular dynamics simulations that allow for the rationalization of experimental observations...
March 15, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28291518/a-novel-expression-system-for-lytic-polysaccharide-monooxygenases
#12
Gaston Courtade, Simone Balzer Le, Gerd Inger Sætrom, Trygve Brautaset, Finn L Aachmann
Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space...
February 14, 2017: Carbohydrate Research
https://www.readbyqxmd.com/read/28288816/a-comparative-approach-to-recombinantly-produce-the-plant-enzyme-horseradish-peroxidase-in-escherichia-coli
#13
Thomas Gundinger, Oliver Spadiut
Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E...
March 11, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28280110/tata-complexes-exhibit-a-marked-change-in-organisation-in-response-to-expression-of-the-tatbc-complex
#14
Sarah Marie Smith, Andrew Yarwood, Roland A Fleck, Colin Robinson, Corinne J Smith
The twin arginine translocation (Tat) system is an integral membrane protein complex that accomplishes the remarkable feat of transporting large, fully-folded polypeptides across the inner membrane of bacteria, into the periplasm. In Escherichia coli Tat is comprised of three membrane proteins: TatA, TatB and TatC. How these proteins arrange themselves in the inner membrane to permit passage of Tat substrates, whilst maintaining membrane integrity, is still poorly understood.  TatA is the most abundant component of this complex and facilitates assembly of the transport mechanism...
March 9, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28272446/critical-role-of-quorum-sensing-dependent-glutamate-metabolism-in-homeostatic-osmolality-and-outer-membrane-vesiculation-in-burkholderia-glumae
#15
Yongsung Kang, Eunhye Goo, Jinwoo Kim, Ingyu Hwang
Metabolic homeostasis in cooperative bacteria is achieved by modulating primary metabolism in a quorum sensing (QS)-dependent manner. A perturbed metabolism in QS mutants causes physiological stress in the rice bacterial pathogen Burkholderia glumae. Here, we show that increased bacterial osmolality in B. glumae is caused by unusually high cellular concentrations of glutamate and betaine generated by QS deficiencies. QS negatively controls glutamate uptake and the expression of genes involved in the glutamine synthetase and glutamine oxoglutarate aminotransferase cycles...
March 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28267156/maltose-binding-protein-effectively-stabilizes-the-partially-closed-conformation-of-the-atp-binding-cassette-transporter-malfgk2
#16
Jingwei Weng, Shuo Gu, Xin Gao, Xuhui Huang, Wenning Wang
Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called "pretranslocation" ("pre-T") state with a partially closed conformation suggest that the formation of this MalE-stabilized intermediate state is a key step leading to the outward-facing catalytic state. On the contrary, crosslinking and fluorescence studies suggest that ATP binding alone is sufficient to promote the outward-facing catalytic state, thereby doubting the role of MalE binding...
March 7, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28264993/going-outside-the-tonb-box-identification-of-novel-fepa-tonb-interactions-in-vivo
#17
Michael G Gresock, Kathleen Postle
In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from protonmotive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta-barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158-162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork...
March 6, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28263038/divide-and-conquer-the-pseudomonas-aeruginosa-two-component-hybrid-sags-enables-biofilm-formation-and-recalcitrance-of-biofilm-cells-to-antimicrobial-agents-via-distinct-regulatory-circuits
#18
Olga E Petrova, Kajal Gupta, Julie Liao, James S Goodwine, Karin Sauer
The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbors a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains...
March 6, 2017: Environmental Microbiology
https://www.readbyqxmd.com/read/28259516/engineering-a-switch-based-biosensor-for-arginine-using-a-thermotoga-maritima-periplasmic-binding-protein
#19
Teraya Donaldson, Luisa Iozzino, Lindsay J Deacon, Hilbert Billones, Alessio Ausili, Sabato D'Auria, Jonathan D Dattelbaum
The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine...
March 1, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28258547/-1-h-15-n-and-13-c-resonance-assignments-and-secondary-structure-of-pulg-the-major-pseudopilin-from-klebsiella-oxytoca-type-2-secretion-system
#20
Aracelys López-Castilla, Bruno Vitorge, Léa Khoury, Nelly Morellet, Olivera Francetic, Nadia Izadi-Pruneyre
Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery...
March 3, 2017: Biomolecular NMR Assignments
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