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Recombinant protein

Bryan A Johnson, Heather L Aloor, Cary A Moody
Human papillomaviruses (HPV) exhibit constitutive activation of ATM and ATR DNA damage response (DDR) pathways, which are required for productive viral replication. Expression of HPV31 E7 alone is sufficient to activate the DDR through an unknown mechanism. Here, we demonstrate that the E7 Rb binding domain is required to increase levels of many DDR proteins, including ATM, Chk2, Chk1, the MRN components MRE11, Rad50, and NBS1, as well as the homologous recombination repair proteins BRCA1 and Rad51. Interestingly, we have found that the increase in these DNA repair proteins does not occur solely at the level of transcription, but that E7 broadly increases the half-life of these DDR factors, a phenotype that is lost in the E7 Rb binding mutant...
October 19, 2016: Virology
Nicole Groh, Christian Seutter von Loetzen, Brinda Subbarayal, Christian Möbs, Lothar Vogel, Andreas Hoffmann, Kay Fötisch, Anna Koutsouridou, Stefanie Randow, Elke Völker, Andreas Reuter, Paul Rösch, Stefan Vieths, Wolfgang Pfützner, Barbara Bohle, Dirk Schiller
BACKGROUND: Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. OBJECTIVE: We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. METHODS: 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed...
October 22, 2016: Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology
Anthony L Cunningham, Nathalie Garçon, Oberdan Leo, Leonard R Friedland, Richard Strugnell, Béatrice Laupèze, Mark Doherty, Peter Stern
In the 21st century, an array of microbiological and molecular allow antigens for new vaccines to be specifically identified, designed, produced and delivered with the aim of optimising the induction of a protective immune response against a well-defined immunogen. New knowledge about the functioning of the immune system and host pathogen interactions has stimulated the rational design of vaccines. The design toolbox includes vaccines made from whole pathogens, protein subunits, polysaccharides, pathogen-like particles, use of viral/bacterial vectors, plus adjuvants and conjugation technology to increase and broaden the immune response...
October 18, 2016: Vaccine
Wei Shen, Ying Xue, Yiqi Liu, Chuixing Kong, Xiaolong Wang, Mengmeng Huang, Menghao Cai, Xiangshan Zhou, Yuanxing Zhang, Mian Zhou
BACKGROUND: As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P AOX1 ) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in P AOX1 activation or repression by different carbon sources may be promising targets...
October 21, 2016: Microbial Cell Factories
Sha Xu, Ge-Yuan Zhang, Huijie Zhang, Toshihiko Kitajima, Hideki Nakanishi, Xiao-Dong Gao
BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. RESULTS: In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain...
October 21, 2016: Microbial Cell Factories
Xiaoxue Tong, Tania Triscari Barberi, Catherine H Botting, Sunil V Sharma, Mark J H Simmons, Tim W Overton, Rebecca J M Goss
BACKGROUND: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis...
October 21, 2016: Microbial Cell Factories
Ryota Saito, Maiko Hoshi, Akihiro Kato, Chikako Ishikawa, Toshiya Komatsu
A number of (Z)-4-arylmethylene-1H-imidazol-5(4H)-ones, which are related to the fluorescent chromophore of the Aequorea green fluorescent protein (GFP), have been synthesized and evaluated their in vitro inhibitory activity against recombinant human aldose reductase for the first time. The GFP chromophore model 1a, with a p-hydroxy group on the 4-benzylidene and a carboxymethyl group on the N1 position, exhibited strong bioactivity with an IC50 value of 0.36 μM. This efficacy is higher than that of sorbinil, a known highly potent aldose reductase inhibitor...
October 8, 2016: European Journal of Medicinal Chemistry
Maria Chatzinikolaidou, Charalampos Pontikoglou, Konstantina Terzaki, Maria Kaliva, Athanasia Kalyva, Eleni Papadaki, Maria Vamvakaki, Maria Farsari
The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200μm increased significantly after 10days in culture, however without significant differences among them...
October 13, 2016: Colloids and Surfaces. B, Biointerfaces
Jialin Zhang, Wenxing Liu, Weiye Chen, Cuicui Li, Meimei Xie, Zhigao Bu
From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations...
2016: PloS One
Qian Lu, Jihong Wang, Junshu Jiang, Shengnan Wang, Qilan Jia, Yue Wang, Weiping Li, Qin Zhou, Li Lv, Qingwei Li
BACKGROUND: The RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model...
2016: PloS One
Lasse van Wijlick, René Geissen, Jessica S Hilbig, Quentin Lagadec, Pilar D Cantero, Eugen Pfeifer, Mateusz Juchimiuk, Sven Kluge, Stephan Wickert, Paula Alepuz, Joachim F Ernst
In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation...
October 2016: PLoS Genetics
Philippe Lefrançois, Beth Rockmill, Pingxing Xie, G Shirleen Roeder, Michael Snyder
During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required...
October 2016: PLoS Genetics
Kerry A Geiler-Samerotte, Yuan O Zhu, Benjamin E Goulet, David W Hall, Mark L Siegal
The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90's ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast...
October 2016: PLoS Biology
Tao Peng, Howard C Hang
Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells...
October 21, 2016: Journal of the American Chemical Society
Ji Luo, Qingyang Liu, Kunihiko Morihiro, Alexander Deiters
Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest...
November 2016: Nature Chemistry
Benoîte Bourdin, Emilie Segura, Marie-Philippe Tétreault, Sylvie Lesage, Lucie Parent
Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1...
September 28, 2016: Journal of Visualized Experiments: JoVE
Chen Luo, Lu-Ting Yu, Meng-Qi Yang, Xiang Li, Zhi-Yuan Zhang, Martin O Alfred, Jun-Li Liu, Min Wang
Regenerating genes (Reg) have been found during the search for factors involved in pancreatic islet regeneration. Our recent study discovered that pancreatic β-cell-specific overexpression of Reg3β protects against streptozotocin (Stz) -induced diabetes in mice. To investigate its potential roles in the treatment of diabetes, we produced a recombinant Reg3β protein and provided evidence that it is active in promoting islet β-cell survival against Stz- triggered cell death. Though ineffective in alleviating preexisting diabetes, pretreatment of recombinant Reg3β was capable of minimizing the Stz-induced hyperglycemia and weight loss, by preserving serum and pancreatic insulin levels, and islet β-cell mass...
October 21, 2016: Scientific Reports
Hongyou Yu, Corey S Moran, Alexandra F Trollope, Lynn Woodward, Robert Kinobe, Catherine M Rush, Jonathan Golledge
Angiogenesis and inflammation are implicated in aortic aneurysm and atherosclerosis and regulated by angiopoietin-2 (Angpt2). The effect of Angpt2 administration on experimental aortic aneurysm and atherosclerosis was examined. Six-month-old male apolipoprotein E deficient (ApoE(-/-)) mice were infused with angiotensin II (AngII) and administered subcutaneous human Fc-protein (control) or recombinant Angpt2 (rAngpt2) over 14 days. Administration of rAngpt2 significantly inhibited AngII-induced aortic dilatation and rupture of the suprarenal aorta (SRA), and development of atherosclerosis within the aortic arch...
October 21, 2016: Scientific Reports
Muhammad Rafehi, Joachim C Burbiel, Isaac Y Attah, Aliaa Abdelrahman, Christa E Müller
The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties...
October 20, 2016: Purinergic Signalling
Sher Bahadur Poudel, Govinda Bhattarai, Jae-Hwan Kim, Sung-Ho Kook, Young-Kwon Seo, Young-Mi Jeon, Jeong-Chae Lee
Fibroblast growth factor 7 (FGF7) plays an important role in regulating the proliferation, migration, and differentiation of cells. However, the role of FGF7 in bone formation is not yet fully understood. We examined the effect of FGF7 on bone formation using a rat model of mandible defects. Rats underwent mandible defect surgery and then either scaffold treatment alone (control group) or FGF7-impregnated scaffold treatment (FGF7 group). Micro-CT and histological analyses revealed that the FGF7 group exhibited greater bone formation than did the control group 10 weeks after surgery...
October 20, 2016: Journal of Bone and Mineral Metabolism
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