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https://www.readbyqxmd.com/read/28231319/development-of-a-keratinase-activity-assay-using-recombinant-chicken-feather-keratin-substrates
#1
Hyeon-Su Jin, Seon Yeong Park, Kyungmin Kim, Yong-Jik Lee, Gae-Won Nam, Nam Joo Kang, Dong-Woo Lee
Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins...
2017: PloS One
https://www.readbyqxmd.com/read/28231271/applying-antibody-sensitive-hypervariable-region-1-deleted-hepatitis-c-virus-to-the-study-of-escape-pathways-of-neutralizing-human-monoclonal-antibody-ar5a
#2
Rodrigo Velázquez-Moctezuma, Mansun Law, Jens Bukh, Jannick Prentoe
Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3-4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope...
February 23, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28230977/extracellular-self-assembly-of-functional-and-tunable-protein-conjugates-from-bacillus-subtilis
#3
Charlie Gilbert, Mark Howarth, Colin R Harwood, Tom Ellis
The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically-encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis...
February 23, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28230975/tuning-of-recombinant-protein-expression-in-escherichia-coli-by-manipulating-transcription-translation-initiation-rates-and-incorporation-of-non-canonical-amino-acids
#4
Orr Schlesinger, Yonatan Chemla, Mathias Heltberg, Eden Ozer, Ryan Marshall, Vincent Noireaux, Mogens Hogh Jensen, Lital Alfonta
Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression...
February 23, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28230529/single-molecule-fret-unveils-induced-fit-mechanism-for-substrate-selectivity-in-flap-endonuclease-1
#5
Fahad Rashid, Paul D Harris, Manal S Zaher, Mohamed A Sobhy, Luay I Joudeh, Chunli Yan, Hubert Piwonski, Susan E Tsutakawa, Ivaylo Ivanov, John A Tainer, Satoshi Habuchi, Samir M Hamdan
Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage...
February 23, 2017: ELife
https://www.readbyqxmd.com/read/28230321/disruption-of-genes-involved-in-corvet-complex-leads-to-enhanced-secretion-of-heterologous-carboxylesterase-only-in-protease-deficient-pichia-pastoris
#6
Lukas Marsalek, Clemens Gruber, Friedrich Altmann, Markus Aleschko, Diethard Mattanovich, Brigitte Gasser, Verena Puxbaum
The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work we have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET tethering complex at the early stages of endosomal sorting...
February 23, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28230059/synergistic-effects-of-dimethyloxallyl-glycine-and-recombinant-human-bone-morphogenetic-protein-2-on-repair-of-critical-sized-bone-defects-in-rats
#7
Xin Qi, Yang Liu, Zhen-Yu Ding, Jia-Qing Cao, Jing-Huan Huang, Jie-Yuan Zhang, Wei-Tao Jia, Jing Wang, Chang-Sheng Liu, Xiao-Lin Li
In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold...
February 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28229932/csn5-jab1-suppresses-the-wnt-inhibitor-dkk1-in-colorectal-cancer-cells
#8
Sandra Jumpertz, Thomas Hennes, Yaw Asare, Anke K Schütz, Jürgen Bernhagen
The COP9 signalosome (CSN) is a multi-protein complex that is highly conserved in eukaryotes. Due to its regulatory impact on processes such as cell cycle, DNA damage response and apoptosis, the CSN is essential for mammalian cells. One of the best-studied functions of the CSN is the deNEDDylation of cullin-RING ligases (CRLs) via its catalytically active subunit CSN5/JAB1, thereby triggering the degradation of various target proteins. CSN5 was found to be overexpressed in many human cancer entities, including colon adenocarcinoma...
February 13, 2017: Cellular Signalling
https://www.readbyqxmd.com/read/28229548/oligofructose-decreases-serum-lipopolysaccharide-and-plasminogen-activator-inhibitor-1-in-adults-with-overweight-obesity
#9
Jill A Parnell, Teja Klancic, Raylene A Reimer
OBJECTIVE: To determine the effect of prebiotic supplementation on metabolic endotoxemia and systemic inflammation in adults with overweight and obesity. METHODS: Samples from a previously conducted randomized, double-blind, placebo-controlled trial were used for analysis. Participants were randomized to 21 g of oligofructose (n = 20; BMI 30.4 kg/m(2) ) or a maltodextrin placebo (n = 17; BMI 29.5 kg/m(2) ) for 12 weeks. A total of 37 participants had samples available for the current analysis...
March 2017: Obesity
https://www.readbyqxmd.com/read/28229435/design-and-pharmacodynamics-of-recombinant-nz2114-histidine-mutants-with-improved-activity-against-methicillin-resistant-staphylococcus-aureus
#10
Huixian Chen, Ruoyu Mao, Da Teng, Xiumin Wang, Ya Hao, Xingjun Feng, Jianhua Wang
NZ2114 is a promising candidate for therapeutic application owing to its potent activity to Staphylococcus aureus. Our objective was to identify NZ2114 derivatives with improved activity through substitution of His16 and His18 with residues Arginine and Lysine. Eight mutants were designed and expressed in Pichia pastoris X-33 via pPICZαA. Five of them exhibited strong antimicrobial activity against S. aureus at low minimal inhibitory concentrations (MICs) of 0.057-0.454 μM. Among them, H1, H2, and H3 showed ideal pharmacodynamic effects on methicillin-resistant S...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28229208/trichoderma-reesei-xylanase-5-is-defective-in-the-reference-strain-qm6a-but-functional-alleles-are-present-in-other-wild-type-strains
#11
Jonas Ramoni, Martina Marchetti-Deschmann, Verena Seidl-Seiboth, Bernhard Seiboth
Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5...
February 22, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28228925/what-can-mathematical-modelling-say-about-cho-metabolism-and-protein-glycosylation
#12
REVIEW
Sarah N Galleguillos, David Ruckerbauer, Matthias P Gerstl, Nicole Borth, Michael Hanscho, Jürgen Zanghellini
Chinese hamster ovary cells have been in the spotlight for process optimization in recent years, due to being the major, long established cell factory for the production of recombinant proteins. A deep, quantitative understanding of CHO metabolism and mechanisms involved in protein glycosylation has proven to be attainable through the development of high throughput technologies. Here we review the most notable accomplishments in the field of modelling CHO metabolism and protein glycosylation.
2017: Computational and Structural Biotechnology Journal
https://www.readbyqxmd.com/read/28228639/grin1-mutation-associated-with-intellectual-disability-alters-nmda-receptor-trafficking-and-function
#13
Wenjuan Chen, Christine Shieh, Sharon A Swanger, Anel Tankovic, Margaret Au, Marianne McGuire, Michele Tagliati, John M Graham, Suneeta Madan-Khetarpal, Stephen F Traynelis, Hongjie Yuan, Tyler Mark Pierson
N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties...
February 23, 2017: Journal of Human Genetics
https://www.readbyqxmd.com/read/28228592/functional-relevance-of-the-n-terminal-domain-of-pseudorabies-virus-envelope-glycoprotein-h-and-its-interaction-with-glycoprotein-l
#14
Melina Vallbracht, Sascha Rehwaldt, Barbara G Klupp, Thomas C Mettenleiter, Walter Fuchs
Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein gB and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV) phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH, gH(B4...
February 22, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28228545/ablation-of-rnai-and-retrotransposons-accompany-acquisition-and-evolution-of-transposases-to-heterochromatin-protein-cenpb
#15
Udita Upadhyay, Suchita Srivastava, Indu Khatri, Jagpreet Singh Nanda, Srikrishna Subramanian, Amit Arora, Jagmohan Singh
Inactivation of retrotransposons is accompanied by the emergence of CENPB (centromere binding protein-B) in Schizosaccharomyces as well as in metazoans. The RNAi-induced Transcriptional Silencing (RITS) Complex, comprising Chp1 (chromodomain protein-1), Tas3 (protein with unknown function) and Ago1 (Argonaute), plays an important role in RNAi-mediated heterochromatinization. We find that while the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in S. cryophilus and S...
February 22, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28228534/topbp1-dpb11-plays-a-conserved-role-in-homologous-recombination-dna-repair-through-the-coordinated-recruitment-of-53bp1-rad9
#16
Yi Liu, José Renato Cussiol, Diego Dibitetto, Jennie Rae Sims, Shyam Twayana, Robert Samuel Weiss, Raimundo Freire, Federica Marini, Achille Pellicioli, Marcus Bustamante Smolka
Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1(Dpb11) has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold...
February 22, 2017: Journal of Cell Biology
https://www.readbyqxmd.com/read/28228481/a-helical-bundle-in-the-n-terminal-domain-of-the-blm-helicase-mediates-dimer-and-potentially-hexamer-formation
#17
Jing Shi, Wei-Fei Chen, Bo Zhang, San-Hong Fan, Xia Ai, Na-Nv Liu, Stephane Rety, Xu-Guang Xi
Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In human, mutations in genes of three members of the RecQ family helicases (blm, wrn and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking...
February 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28228395/antibody-responses-to-zika-virus-infections-in-flavivirus-endemic-environments
#18
Sarah L Keasey, Christine L Pugh, Stig M R Jensen, Jessica L Smith, Robert D Hontz, Anna P Durbin, Dawn M Dudley, David H O'Connor, Robert G Ulrich
Zika virus (ZIKV) infections occur in areas where dengue (DENV), West Nile (WNV), yellow fever (YFV), and other viruses of the genus Flavivirus co-circulate. The envelope protein (E) of these closely related flaviviruses induces specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV and YFV infections of humans and non-human primates (NHP)...
February 22, 2017: Clinical and Vaccine Immunology: CVI
https://www.readbyqxmd.com/read/28228321/immunization-of-bighorn-sheep-against-mannheimia-haemolytica-with-a-bovine-herpesvirus-1-vectored-vaccine
#19
Sai A Batra, Sudarvili Shanthalingam, Gaetano Donofrio, Gary J Haldorson, Shafiqul Chowdhury, Stephen N White, Subramaniam Srikumaran
Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible...
February 19, 2017: Vaccine
https://www.readbyqxmd.com/read/28228181/characterization-and-functional-analysis-of-serpin-1-like-gene-from-oak-silkworm-antheraea-pernyi
#20
H M Yu, B J Zhu, Y Sun, G Q Wei, L Wang, C Qian, M Nadeem Abbas, C L Liu
Serpins are a broadly distributed family of proteases found in various organisms that play an important role in regulating the immune response. Here, we identified a serpin-1 gene from Antheraea pernyi that encodes a 279 amino acid protein with a molecular weight of 30.8 kDa. We expressed the recombinant Ap-serpin-1 protein in Escherichia coli and used the purified protein to prepare rabbit anti-Ap-serpin-1 polyclonal antibodies. We calculated the enzyme-linked immunosorbent assay titer of the antibody as 1:128000...
February 23, 2017: Bulletin of Entomological Research
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