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Protein engineering

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https://www.readbyqxmd.com/read/28432956/introduction-of-n-cadherin-binding-motif-to-alginate-hydrogels-for-controlled-stem-cell-differentiation
#1
Jae Won Lee, Hyoseok An, Kuen Yong Lee
Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif...
April 9, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28432439/characterization-of-a-functional-recombinant-human-creatine-kinase-mb-isoenzyme-prepared-by-tandem-affinity-purification-from-escherichia-coli
#2
Lihui Zou, Wen Su, Meng Wang, Wei Huang, Haijian Zhao, Enyi Zhang, Junhua Jin, Hongtao Xu, Fei Xiao
Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories...
April 21, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28432329/affinity-biosensors-using-recombinant-native-membrane-proteins-displayed-on-exosomes-application-to-botulinum-neurotoxin-b-receptor
#3
Richard Desplantes, Christian Lévêque, Benjamin Muller, Manuela Lotierzo, Géraldine Ferracci, Michel Popoff, Michael Seagar, Robert Mamoun, Oussama El Far
The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes...
April 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28432282/loss-of-function-myostatin-mutation-increases-insulin-sensitivity-and-browning-of-white-fat-in-meishan-pigs
#4
Chunbo Cai, Lili Qian, Shengwang Jiang, Youde Sun, Qingqing Wang, Dezun Ma, Gaojun Xiao, Biao Li, Shanshan Xie, Ting Gao, Yaoxing Chen, Jie Liu, Xiaorong An, Wentao Cui, Kui Li
Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn -/- ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn -/- pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue...
April 4, 2017: Oncotarget
https://www.readbyqxmd.com/read/28432100/casein-kinase-i-isoform-hrr25-is-a-negative-regulator-of-haa1-in-the-weak-acid-stress-response-pathway-in-saccharomyces-cerevisiae
#5
Morgan E Collins, Joshua J Black, Zhengchang Liu
Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1-target genes, and that Hrr25 interacts with Haa1...
April 21, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28431257/integrated-antimicrobial-and-antifouling-ultrafiltration-membrane-by-surface-grafting-peo-and-n-chloramine-functional-groups
#6
Shuhua Hou, Jialin Xing, Xue Dong, Jifu Zheng, Shenghai Li
Ultrafiltration membranes with integrated antimicrobial and antifouling properties were fabricated using an engineering thermoplastic (carboxylated cardopoly(aryl ether ketone, PEK-COOH). Different molecular weights of PEO (Mw: 120, 350, 550) were grafted to the PEK-COOH membrane surface via EDC/NHS methodology. N-chloramine modified membranes then were prepared by simple exposure to dilute sodium hypochlorite solution. The surface grafting processes were all performed in water (i.e. without organic solvent)...
April 11, 2017: Journal of Colloid and Interface Science
https://www.readbyqxmd.com/read/28431251/tunable-expression-tools-enable-single-cell-strain-distinction-in-the-gut-microbiome
#7
Weston R Whitaker, Elizabeth Stanley Shepherd, Justin L Sonnenburg
Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut...
April 20, 2017: Cell
https://www.readbyqxmd.com/read/28430426/the-rosetta-all-atom-energy-function-for-macromolecular-modeling-and-design
#8
Rebecca Faye Alford, Andrew Leaver-Fay, Jeliazko R Jeliazkov, Matthew J O'Meara, Frank P DiMaio, Hahnbeom Park, Maxim V Shapovalov, P Douglas Renfrew, Vikram Khipple Mulligan, Kalli Kappel, Jason W Labonte, Michael Steven Pacella, Richard Bonneau, Philip Bradley, Roland L Dunbrack, Rhiju Das, David Baker, Brian Kuhlman, Tanja Kortemme, Jeffrey J Gray
Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta's success is the energy function: a model parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta Energy Function, REF15...
April 21, 2017: Journal of Chemical Theory and Computation
https://www.readbyqxmd.com/read/28430399/targeting-conformational-activation-of-cdk2-kinase
#9
Morgan Pellerano, Sergey Tcherniuk, Corine Perals, Thi Nhu Ngoc Van, Elsa Garcin, Florence Mahuteau-Betzer, Marie-Paule Teulade-Fichou, May C Morris
Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation...
April 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28430190/tuning-underwater-adhesion-with-cation-%C3%AF-interactions
#10
Matthew A Gebbie, Wei Wei, Alex M Schrader, Thomas R Cristiani, Howard A Dobbs, Matthew Idso, Bradley F Chmelka, J Herbert Waite, Jacob N Israelachvili
Cation-π interactions drive the self-assembly and cohesion of many biological molecules, including the adhesion proteins of several marine organisms. Although the origin of cation-π bonds in isolated pairs has been extensively studied, the energetics of cation-π-driven self-assembly in molecular films remains uncharted. Here we use nanoscale force measurements in combination with solid-state NMR spectroscopy to show that the cohesive properties of simple aromatic- and lysine-rich peptides rival those of the strong reversible intermolecular cohesion exhibited by adhesion proteins of marine mussel...
May 2017: Nature Chemistry
https://www.readbyqxmd.com/read/28430023/harnessing-effector-triggered-immunity-for-durable-disease-resistance
#11
Meixiang Zhang, Gitta Coaker
Genetic control of plant diseases has traditionally included the deployment of single immune receptors with nucleotide-binding leucine-rich repeat (NLR) domain architecture. These NLRs recognize corresponding pathogen effector proteins inside plant cells, resulting in effector-triggered immunity (ETI). Although ETI triggers robust resistance, deployment of single NLRs can be rapidly overcome by pathogen populations within a single or a few growing seasons. In order to generate more durable disease resistance against devastating plant pathogens, a multi-tiered strategy that incorporates stacked NLRs combined with other sources of disease resistance is necessary...
April 21, 2017: Phytopathology
https://www.readbyqxmd.com/read/28429845/following-nature-s-roadmap-folding-factors-from-plasma-cells-led-to-improvements-in-antibody-secretion-in-s-cerevisiae
#12
Essi V Koskela, Jorg C de Ruijter, Alexander D Frey
Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite multiple numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, we demonstrate the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast...
April 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28429809/successful-pegylation-of-hollow-encapsulin-nanoparticles-from-rhodococcus-erythropolis-n771-without-affecting-their-disassembly-and-reassembly-properties
#13
Seiichi Sonotaki, Taku Takami, Keiichi Noguchi, Masafumi Odaka, Masafumi Yohda, Yoshihiko Murakami
We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent...
April 21, 2017: Biomaterials Science
https://www.readbyqxmd.com/read/28429713/gating-of-tonb-dependent-transporters-by-substrate-specific-forced-remodelling
#14
Samuel J Hickman, Rachael E M Cooper, Luca Bellucci, Emanuele Paci, David J Brockwell
Membrane proteins play vital roles in inside-out and outside-in signal transduction by responding to inputs that include mechanical stimuli. Mechanical gating may be mediated by the membrane or by protein(s) but evidence for the latter is scarce. Here we use force spectroscopy, protein engineering and bacterial growth assays to investigate the effects of force on complexes formed between TonB and TonB-dependent transporters (TBDT) from Gram-negative bacteria. We confirm the feasibility of protein-only mediated mechanical gating by demonstrating that the interaction between TonB and BtuB (a TBDT) is sufficiently strong under force to create a channel through the TBDT...
April 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28429326/wax-ester-fermentation-and-its-application-for-biofuel-production
#15
Hiroshi Inui, Takahiro Ishikawa, Masahiro Tamoi
In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28428823/combining-manipulation-of-transcription-factors-and-overexpression-of-the-target-genes-to-enhance-lignocellulolytic-enzyme-production-in-penicillium-oxalicum
#16
Liwei Gao, Zhonghai Li, Chengqiang Xia, Yinbo Qu, Meng Liu, Piao Yang, Lele Yu, Xin Song
BACKGROUND: Lignocellulolytic enzymes are the main enzymes to saccharify lignocellulose from renewable plant biomass in the bio-based economy. The production of these enzymes is transcriptionally regulated by multiple transcription factors. We previously engineered Penicillium oxalicum for improved cellulase production via manipulation of three genes in the cellulase expression regulatory network. However, the potential of combinational engineering of multiple regulators and their targets at protein abundance and activity levels has not been fully explored...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28428821/genome-wide-search-for-candidate-genes-for-yeast-robustness-improvement-against-formic-acid-reveals-novel-susceptibility-trk1-and-positive-regulators-and-resistance-haa1-regulon-determinants
#17
Sílvia F Henriques, Nuno P Mira, Isabel Sá-Correia
BACKGROUND: Formic acid is an inhibitory compound present in lignocellulosic hydrolysates. Understanding the complex molecular mechanisms underlying Saccharomyces cerevisiae tolerance to this weak acid at the system level is instrumental to guide synthetic pathway engineering for robustness improvement of industrial strains envisaging their use in lignocellulosic biorefineries. RESULTS: This study was performed to identify, at a genome-wide scale, genes whose expression confers protection or susceptibility to formic acid, based on the screening of a haploid deletion mutant collection to search for these phenotypes in the presence of 60, 70 and 80 mM of this acid, at pH 4...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28428810/recent-achievements-obtained-by-chloroplast-transformation
#18
REVIEW
Muhamed Adem, Dereje Beyene, Tileye Feyissa
Chloroplasts play a great role for sustained wellbeing of life on the planet. They have the power and raw materials that can be used as sophisticated biological factories. They are rich in energy as they have lots of pigment-protein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in minerals imported from the plant cell. Chloroplast genome transformation offers multiple advantages over nuclear genome which among others, include: integration of the transgene via homologus recombination that enables to eliminate gene silencing and position effect, higher level of transgene expression resulting into higher accumulations of foreign proteins, and significant reduction in environmental dispersion of the transgene due to maternal inheritance which helps to minimize the major critic of plant genetic engineering...
2017: Plant Methods
https://www.readbyqxmd.com/read/28428022/cloning-and-identification-of-a-novel-steroid-11%C3%AE-hydroxylase-gene-from-absidia-coerulea
#19
Ruijie Wang, Pengcheng Sui, Xiangjiang Hou, Tong Cao, Longgang Jia, Fuping Lu, Suren Singh, Zhengxiang Wang, Xiaoguang Liu
Steroid 11-hydroxylation by filamentous fungi is a major route for industrial scale production of key intermediates in the synthesis of steroid drugs. Although it is well established that enzymes involved in fungal hydroxylation of steroids are cytochrome P450s (CYP), few fungal steroid hydroxylase genes have been identified. In this study, we identified a novel 11α-hydroxylase gene CYP5311B1 from Absidia coerulea AS3.65 by a combination of transcriptome sequencing, Real-Time qRT-PCR and heterologous expression in Pichia pastoris...
April 17, 2017: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28427825/molecular-mechanisms-of-the-yeast-adaptive-response-and-tolerance-to-stresses-encountered-during-ethanol-fermentation
#20
REVIEW
Choowong Auesukaree
During ethanol fermentation, yeast cells encounter various stresses including sugar substrates-induced high osmolarity, increased ethanol concentration, oxygen metabolism-derived reactive oxygen species (ROS), and elevated temperature. To cope with these fermentation-associated stresses, appropriate adaptive responses are required to prevent stress-induced cellular dysfunctions and to acquire stress tolerances. This review will focus on the cellular effects of these stresses, molecular basis of the adaptive response to each stress, and the cellular mechanisms contributing to stress tolerance...
April 17, 2017: Journal of Bioscience and Bioengineering
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