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https://www.readbyqxmd.com/read/28922417/lingering-single-strand-breaks-trigger-rad51-independent-homology-directed-repair-of-collapsed-replication-forks-in-the-polynucleotide-kinase-phosphatase-mutant-of-fission-yeast
#1
Arancha Sanchez, Mariana C Gadaleta, Oliver Limbo, Paul Russell
The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5'-phosphate and 3'-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1Δ) cells of Schizosaccharomyces pombe...
September 18, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28922413/exploitation-of-stable-nanostructures-based-on-the-mouse-polyomavirus-for-development-of-a-recombinant-vaccine-against-porcine-circovirus-2
#2
Martin Fraiberk, Michaela Hájková, Magdaléna Krulová, Martina Kojzarová, Alena Drda Morávková, Ivan Pšikal, Jitka Forstová
The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens...
2017: PloS One
https://www.readbyqxmd.com/read/28922357/analysis-of-nuclear-and-organellar-genomes-of-plasmodium-knowlesi-in-humans-reveals-ancient-population-structure-and-recent-recombination-among-host-specific-subpopulations
#3
Ernest Diez Benavente, Paola Florez de Sessions, Robert W Moon, Anthony A Holder, Michael J Blackman, Cally Roper, Christopher J Drakeley, Arnab Pain, Colin J Sutherland, Martin L Hibberd, Susana Campino, Taane G Clark
The macaque parasite Plasmodium knowlesi is a significant concern in Malaysia where cases of human infection are increasing. Parasites infecting humans originate from genetically distinct subpopulations associated with the long-tailed (Macaca fascicularis (Mf)) or pig-tailed macaques (Macaca nemestrina (Mn)). We used a new high-quality reference genome to re-evaluate previously described subpopulations among human and macaque isolates from Malaysian-Borneo and Peninsular-Malaysia. Nuclear genomes were dimorphic, as expected, but new evidence of chromosomal-segment exchanges between subpopulations was found...
September 18, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28921896/evaluating-potential-risks-of-food-allergy-and-toxicity-of-soy-leghemoglobin-expressed-in-pichia-pastoris
#4
Yuan Jin, Xiaoyun He, Kwame Andoh-Kumi, Rachel Z Fraser, Mei Lu, Richard E Goodman
SCOPE: The Soybean (Glycine max) leghemoglobin c2 (LegHb) gene was introduced into Pichia pastoris yeast for sustainable production of a heme-carrying protein, for organoleptic use in plant-based meat. The potential allergenicity and toxicity of LegHb and 17 Pichia host-proteins each representing ≥1% of total protein in production batches were evaluated by literature review, bioinformatics sequence comparisons to known allergens or toxins, and in vitro pepsin digestion. METHODS AND RESULTS: Literature searches found no evidence of allergenicity or toxicity for these proteins...
September 18, 2017: Molecular Nutrition & Food Research
https://www.readbyqxmd.com/read/28921867/polymer-cancerostatics-targeted-with-an-antibody-fragment-bound-via-a-coiled-coil-motif-in-vivo-therapeutic-efficacy-against-murine-bcl1-leukemia
#5
Michal Pechar, Robert Pola, Olga Janoušková, Irena Sieglová, Vlastimil Král, Milan Fábry, Barbora Tomalová, Marek Kovář
A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein...
September 15, 2017: Macromolecular Bioscience
https://www.readbyqxmd.com/read/28921534/exploring-cellular-behaviour-under-transient-gene-expression-and-its-impact-on-mab-productivity-and-fc-glycosylation
#6
Si Nga Sou, Ken Lee, Kalpana Nayyar, Karen M Polizzi, Christopher Sellick, Cleo Kontoravdi
Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently-expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921455/construction-of-an-hrp-streptavidin-bound-antigen-and-its-application-in-an-elisa-for-porcine-circovirus-2-antibodies
#7
Meng Ge, Run-Cheng Li, Tailong Qu, Wenjie Gong, Xing-Long Yu, Changchun Tu
A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity...
September 18, 2017: AMB Express
https://www.readbyqxmd.com/read/28921441/purification-and-autoactivation-method-for-recombinant-coagulation-factor-vii
#8
Vladimir Granovski, Marcela C C Freitas, Mario Soares Abreu-Neto, Dimas T Covas
Recombinant coagulation factor VII is a very important and complex protein employed for treatment of hemophiliac patients (hemophilia A/B) who develop inhibitors antibodies to conventional treatments (FVIII and FIX). The rFVII is a glycosylated molecule and circulates in plasma as zymogen of 50 kDa. When activated the molecule is cleaved to 20-30 kDa and has a half-life of about 3 h, needing to be processed fast and efficiently until freeze-drying. Here, we describe a very simple and fast purification sequence for rFVII using affinity FVII Select resin and a dialysis system that can be easily scaled up...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921440/microplate-based-method-for-high-throughput-screening-hts-of-chromatographic-conditions-studies-for-recombinant-protein-purification
#9
Rimenys J Carvalho, Thayana A Cruz
High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921439/purification-method-for-recombinant-hg-csf-by-affinity-chromatography
#10
Bruna Samham Archangelo, Elisa Maria de Sousa Russo
The human granulocytic colony-stimulating factor (hG-CSF) acts mainly by promoting the maturation of granulocytes and stimulating their phagocytic and chemotactic activity. It has been used in the treatment of many diseases, in particular in neutropenic conditions. Here, we describe the purification process of the recombinant protein hG-CSF expressed in Pichia pastoris. The protein purification proved to be efficient using the nickel affinity chromatography method described in this chapter.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921438/purification-methods-for-recombinant-factor-viii-expressed-in-human-liver-sk-hep-cells
#11
Vladimir Granovski, Mario Soares Abreu-Neto, Dimas Tadeu Covas
Coagulation factor VIII is one of the largest proteins attempted to be expressed in recombinant form. A very complex and labile protein which has a very short half-live and need a fast and efficient purification chain. Here, we describe a simple purification sequence using multimodal Capto MMC, affinity FVIII select and ion exchange SP-Fastflow chromatography steps without subjecting the target molecule to mechanical and temperature stress, separating impurities from rFVIII using net charge, hydrophobicity, and affinity of the molecules...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921434/bioreactor-based-production-of-glycoproteins-in-plant-cell-suspension-cultures
#12
Tanja Holland, Johannes Felix Buyel
Recombinant glycoproteins such as monoclonal antibodies have a major impact on modern healthcare systems, e.g., as the active pharmaceutical ingredients in anticancer drugs. A specific glycan profile is often necessary to achieve certain desirable activities, such as the effector functions of an antibody, receptor binding or a sufficient serum half-life. However, many expression systems produce glycan profiles that differ substantially from the preferred form (usually the form found in humans) or produce a diverse array of glycans with a range of in vivo activities, thus necessitating laborious and costly separation and purification processes...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921432/demonstration-scale-high-cell-density-fermentation-of-pichia-pastoris
#13
Wan-Cang Liu, Ping Zhu
Pichia pastoris has been one of the most successful heterologous overexpression systems in generating proteins for large-scale production through high-cell-density fermentation. However, optimizing conditions of the large-scale high-cell-density fermentation for biochemistry and industrialization is usually a laborious and time-consuming process. Furthermore, it is often difficult to produce authentic proteins in large quantities, which is a major obstacle for functional and structural features analysis and industrial application...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921431/cell-free-production-of-protein-biologics-within-24-h
#14
Challise J Sullivan, Erik D Pendleton, John Dresios
Protein biologics have emerged as a safe and effective group of drug products that can be used in a variety of medical disorders and clinical settings, including treatment of orphan diseases, personalized medicine, and point-of-care applications. However, the full potential of protein biologics for such applications will not be realized until there are methods available for rapid and cost-effective production of small scale products for individual needs. Here, we describe a modular and scalable method for rapid and adaptable production of protein-based medical products at small doses...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921430/production-of-recombinant-rabies-virus-glycoprotein-by-insect-cells-in-a-single-use-fixed-bed-bioreactor
#15
Daniella C Ventini-Monteiro, Renato M Astray, Carlos A Pereira
A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921429/strategies-to-suspension-serum-free-adaptation-of-mammalian-cell-lines-for-recombinant-glycoprotein-production
#16
Angelo Luis Caron, Rafael Tagé Biaggio, Kamilla Swiech
Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921428/production-of-recombinant-factor-viii-in-human-cell-lines
#17
Daianne Maciely Carvalho Fantacini, Virgínia Picanço-Castro
Human cell lines can produce recombinant proteins much more similar to their natural counterpart, compared to other mammalian cell lines, reducing potential immunogenic reactions. Recombinant proteins produced in nonhuman cells can have in its structure glycan epitopes, such as Galα1,3-Gal (alpha-Gal) and N-glycolylneuraminic acid (Neu5Gc) residues, that are antigenic to humans and can potentially affect the efficacy of the recombinant product. Therefore, the production of recombinant factor VIII (rFVIII) in human cell lines is a new approach to avoid nonhuman glycosylation...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921426/production-of-full-length-antibody-by-pichia-pastoris
#18
Adam Nylen, Ming-Tang Chen
The methylotrophic yeast Pichia pastoris has become an increasingly popular host for recombinant protein expression in recent times. MRL pioneered a glycoengineered humanized P. pastoris expression system that could produce glycoproteins with glycosylation profiles similar to mammalian systems. Therapeutic glycoproteins produced by the humanized P. pastoris platform have shown comparable folding, stability, and in vitro and in vivo efficacies in preclinical models to their counterparts produced from the CHO cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921424/platforms-for-recombinant-therapeutic-glycoprotein-production
#19
Amanda Mizukami, Angelo Luis Caron, Virgínia Picanço-Castro, Kamilla Swiech
The majority of FDA-approved biology-derived products are recombinant glycoproteins. These proteins have been used for the treatment of several diseases, with numerous products currently approved for clinical use. The choice of the expression system is a key step toward a successful functional protein production, since glycosylation influences yield, pharmacokinetics, biological activity, and immunogenicity. This chapter covers the general aspects of therapeutic recombinant glycoproteins and the platforms that are being employed for their production...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28921304/expression-and-purification-of-an-fgf9-fusion-protein-in-e-coli-and-the-effects-of-the-fgf9-subfamily-on-human-hepatocellular-carcinoma-cell-proliferation-and-migration
#20
Shen Wang, Haipeng Lin, Tiantian Zhao, Sisi Huang, David G Fernig, Nuo Xu, Fenfang Wu, Mi Zhou, Chao Jiang, Haishan Tian
Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity...
September 18, 2017: Applied Microbiology and Biotechnology
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