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Protein engineering

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https://www.readbyqxmd.com/read/29334397/recombinant-phosphatidylserine-binding-nanobodies-for-targeting-of-extracellular-vesicles-to-tumor-cells-a-plug-and-play-approach
#1
Sander A A Kooijmans, Jerney J J M Gitz-Francois, Raymond M Schiffelers, Pieter Vader
Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, manipulation of targeting properties of EVs through engineering of the producer cells can be challenging and time-consuming. As a novel approach to confer tumor targeting properties to isolated EVs, we generated recombinant fusion proteins of nanobodies against the epidermal growth factor receptor (EGFR) fused to phosphatidylserine (PS)-binding domains of lactadherin (C1C2)...
January 15, 2018: Nanoscale
https://www.readbyqxmd.com/read/29334187/surveying-the-landscape-of-optogenetic-methods-for-detection-of-protein-protein-interactions
#2
REVIEW
Matthew D Wiens, Robert E Campbell
Mapping the protein-protein interaction (PPi) landscape is of critical importance to furthering our understanding how cells and organisms function. Optogenetic methods, that is, approaches that utilize genetically encoded fluorophores or fluorogenic enzyme reactions, uniquely enable the visualization of biochemical phenomena in live cells with high spatial and temporal accuracy. Applying optogenetic methods to the detection of PPis requires the engineering of protein-based systems in which an optical signal undergoes a substantial change when the two proteins of interest interact...
January 15, 2018: Wiley Interdisciplinary Reviews. Systems Biology and Medicine
https://www.readbyqxmd.com/read/29334092/synthetic-cells-produce-a-quorum-sensing-chemical-signal-perceived-by-pseudomonas-aeruginosa
#3
Giordano Rampioni, Francesca D'Angelo, Marco Messina, Alessandro Zennaro, Yutetsu Kuruma, Daniela Tofani, Livia Leoni, Pasquale Stano
Recent developments in bottom-up synthetic biology (e.g., lipid vesicle technology integrated with cell-free protein expression systems) allow the generation of semi-synthetic minimal cells (in short, synthetic cells, SCs) endowed with some distinctive capacities of natural cells. In particular, such approaches provide technological tools and conceptual frameworks for the design and engineering of programmable SCs capable of communicating with natural cells by exchanging chemical signals. Here we describe the generation of giant vesicle-based SCs which, via gene expression, synthesize in their aqueous lumen an enzyme that in turn produces a chemical signal...
January 15, 2018: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/29333478/a-general-method-for-intracellular-protein-delivery-through-e-tag-protein-engineering-and-arginine-functionalized-gold-nanoparticles
#4
Rubul Mout, Vincent M Rotello
In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al...
December 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29332574/biomacromolecular-based-fibers-in-nanomedicine-a-combination-of-drug-delivery-and-tissue-engineering
#5
Elham Arkan, Abbas Hemati Azandaryani, Pouran Moradipour, Leila Behbood
BACKGROUND: Biopolymers based materials (polysaccharides, lipids, proteins, and nucleic acids) are one of the basic resources in bio-engineering sciences because of desirable features. Moreover, nanobiotechnology innovates nanomaterial and associated technique in nano medicine (drug delivery and tissue engineering). METHODS: In the nano-medicine, fibers are introduced as a successful biomimetic extracellular matrix scaffolds and drug carrier systems. Electrospinning as a simple and cost-effective technique is used to design nanofibers...
January 12, 2018: Current Pharmaceutical Biotechnology
https://www.readbyqxmd.com/read/29332347/genetically-engineered-bone-marrow-derived-mesenchymal-stem-cells-co-expressing-ifn-%C3%AE-and-il-10-inhibit-hepatocellular-carcinoma-by-modulating-mapk-pathway
#6
Hao Wang, Jun Wang, Xiaolei Shi, Yitao Ding
PURPOSE: One of the major challenges in delivering cytokines for the treatment of hepatocellular carcinoma (HCC) is the mode of delivery. This study hypothesized that genetically engineered bone marrow derived mesenchymal stem cells (BMSCs) co-expressing IFN-γ and IL-10 can serve as a potential therapeutic strategy in the treatment of HCC by inhibiting cell proliferation. METHODS: Male Sprague-Dawley rats (n=5, 200-250 g) for BMSCs isolation and Nude/SCID mice (n=35,12-20g) to develop liver cancer xenograft model were used...
November 2017: Journal of B.U.ON.: Official Journal of the Balkan Union of Oncology
https://www.readbyqxmd.com/read/29330459/a-novel-ultra-stable-monomeric-green-fluorescent-protein-for-direct-volumetric-imaging-of-whole-organs-using-clarity
#7
Daniel J Scott, Natalie J Gunn, Kelvin J Yong, Verena C Wimmer, Nicholas A Veldhuis, Leesa M Challis, Mouna Haidar, Steven Petrou, Ross A D Bathgate, Michael D W Griffin
Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively...
January 12, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29330365/engineered-nanointerfaces-for-microfluidic-isolation-and-molecular-profiling-of-tumor-specific-extracellular-vesicles
#8
Eduardo Reátegui, Kristan E van der Vos, Charles P Lai, Mahnaz Zeinali, Nadia A Atai, Berent Aldikacti, Frederick P Floyd, Aimal H Khankhel, Vishal Thapar, Fred H Hochberg, Lecia V Sequist, Brian V Nahed, Bob S Carter, Mehmet Toner, Leonora Balaj, David T Ting, Xandra O Breakefield, Shannon L Stott
Extracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform (EVHB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EVHB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per μL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods...
January 12, 2018: Nature Communications
https://www.readbyqxmd.com/read/29330185/the-mgte-homolog-fici-acts-as-a-secondary-ferrous-iron-importer-in-shewanella-oneidensis-strain-mr-1
#9
Brittany D Bennett, Kaitlyn E Redford, Jeffrey A Gralnick
The transport of metals into and out of cells is necessary for the maintenance of appropriate intracellular concentrations. Metals are needed for incorporation into metalloproteins but become toxic at higher concentrations. Many metal transport proteins have been discovered in bacteria, including the Mg2+ transporter-E (MgtE) family of passive Mg2+/Co2+ cation-selective channels. Low sequence identity exists between members of the MgtE family, indicating that substrate specificity may differ among MgtE transporters...
January 12, 2018: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29330178/development-of-an-efficient-genome-editing-tool-in-bacillus-licheniformis-using-crispr-cas9-nickase
#10
Kaifeng Li, Dongbo Cai, Zhangqian Wang, Zhili He, Shouwen Chen
Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B...
January 12, 2018: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29330023/unraveling-the-differential-structural-stability-and-dynamics-features-of-t7-endolysin-partially-folded-conformations
#11
Meenakshi Sharma, Dinesh Kumar, Krishna Mohan Poluri
BACKGROUND: Characterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5-3, T7L PF states display differential ANS binding characteristics. METHODS: CD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations...
January 9, 2018: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29329531/chemical-genomic-guided-engineering-of-gamma-valerolactone-tolerant-yeast
#12
Scott Bottoms, Quinn Dickinson, Mick McGee, Li Hinchman, Alan Higbee, Alex Hebert, Jose Serate, Dan Xie, Yaoping Zhang, Joshua J Coon, Chad L Myers, Robert Landick, Jeff S Piotrowski
BACKGROUND: Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast...
January 12, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29329501/sustained-plasma-hepcidin-suppression-and-iron-elevation-by-anticalin-derived-hepcidin-antagonist-in-cynomolgus-monkey
#13
Andreas M Hohlbaum, Hendrik Gille, Stefan Trentmann, Maria Kolodziejczyk, Barbara Rattenstetter, Coby M Laarakkers, Galina Katzmann, Hans Jürgen Christian, Nicole Andersen, Andrea Allersdorfer, Shane A Olwill, Bernd Meibohm, Laurent P Audoly, Dorine W Swinkels, Rachel P L van Swelm
BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here we report the engineering of the human lipocalin-derived, small protein-based Anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin- and hepcidin-specific pharmacokinetic/pharmacodynamic modelling was used to design and select the suitable drug candidate based on half life extension and duration of hepcidin suppression...
January 12, 2018: British Journal of Pharmacology
https://www.readbyqxmd.com/read/29329339/accessibility-and-contribution-to-glucan-masking-of-natural-and-genetically-tagged-versions-of-yeast-wall-protein-1-of-candida-albicans
#14
Bruce L Granger
Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized...
2018: PloS One
https://www.readbyqxmd.com/read/29328338/binding-affinity-prediction-of-nanobody-protein-complexes-by-scoring-of-molecular-dynamics-trajectories
#15
Miguel A Soler, Sara Fortuna, Ario de Marco, Alessandro Laio
Nanobodies offer a viable alternative to antibodies for engineering high affinity binders. Their small size has an additional advantage: it allows exploiting computational protocols for optimizing their biophysical features, such as the binding affinity. The efficient prediction of this quantity is still considered a daunting task especially for modelled complexes. We show how molecular dynamics can successfully assist in the binding affinity prediction of modelled nanobody-protein complexes. The approximate initial configurations obtained by in silico design must undergo large rearrangements before achieving a stable conformation, in which the binding affinity can be meaningfully estimated...
January 12, 2018: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29327641/generation-of-an-arrayed-crispr-cas9-library-targeting-epigenetic-regulators-from-high-content-screens-to-in-vivo-assays
#16
Tristan Henser-Brownhill, Josep Monserrat, Paola Scaffidi
The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function...
January 12, 2018: Epigenetics: Official Journal of the DNA Methylation Society
https://www.readbyqxmd.com/read/29326975/identifying-the-substrate-proteins-of-u-box-e3s-e4b-and-chip-by-orthogonal-ubiquitin-transfer
#17
Karan Bhuripanyo, Yiyang Wang, Xianpeng Liu, Li Zhou, Ruochuan Liu, Duc Duong, Bo Zhao, Yingtao Bi, Han Zhou, Geng Chen, Nicholas T Seyfried, Walter J Chazin, Hiroaki Kiyokawa, Jun Yin
E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins...
January 2018: Science Advances
https://www.readbyqxmd.com/read/29326099/interleukin-18-diagnostically-distinguishes-and-pathogenically-promotes-human-and-murine-macrophage-activation-syndrome
#18
Eric S Weiss, Charlotte Girard-Guyonvarc'h, Dirk Holzinger, Adriana A de Jesus, Zeshan Tariq, Jennifer Picarsic, Eduardo J Schiffrin, Dirk Foell, Alexei A Grom, Sandra Ammann, Stephan Ehl, Tomoaki Hoshino, Raphaela Goldbach-Mansky, Cem Gabay, Scott W Canna
Hemophagocytic Lymphohistiocytosis (HLH) and Macrophage Activation Syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Though profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of Interleukin (IL)-18...
January 11, 2018: Blood
https://www.readbyqxmd.com/read/29325564/engineered-recombinant-protein-products-of-the-avian-paramyxovirus-type-1-nucleocapsid-and-phosphoprotein-genes-for-serological-diagnosis
#19
Na Zhao, Christian Grund, Martin Beer, Timm C Harder
BACKGROUND: Virulent Newcastle disease virus (NDV, avian Avulavirus-1, APMV-1) induces a highly contagious and lethal systemic disease in gallinaceous poultry. APMV-1 antibody detection is used for surveillance and to control vaccination, but is hampered by cross-reactivity to other subtypes of avian Avulaviruses. Data are lacking concerning the applicability of NDV V proteins as differential diagnostic marker to distinguish vaccinated from virus-infected birds (DIVA strategy). METHODS: Full length and C-terminally truncated nucleocapsid (NP) protein, and the unique C-terminal regions of the phospho- (P) and V proteins of the NDV LaSota strain were bacterially expressed as fusion proteins with the multimerization domain of the human C4 binding protein, and used as diagnostic antigens in indirect ELISA...
January 11, 2018: Virology Journal
https://www.readbyqxmd.com/read/29324789/an-l213a-variant-of-%C3%AE-glycosidase-from-sulfolobus-solfataricus-with-increased-%C3%AE-l-arabinofuranosidase-activity-converts-ginsenoside-rc-to-compound-k
#20
Ji-Hyeon Choi, Kyung-Chul Shin, Deok-Kun Oh
Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides...
2018: PloS One
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