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https://www.readbyqxmd.com/read/28806153/expression-purification-and-refolding-of-human-lipocalin-6-and-production-of-a-monoclonal-antibody-against-this-protein
#1
Jiong Chen, Wei Feng, Yue Zhao, Yaqin Li, Fei Zhan
Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies...
August 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28806151/establishment-of-a-monoclonal-antibody-against-chgilz
#2
Zhiyuan He, Yongqiang Wang
The chicken glucocorticoid-induced leucine zipper (chGILZ) participates in the inflammation of avian immunosuppressive diseases. We aimed to establish a monoclonal antibody (MAb) against chGILZ and to investigate its distribution in clinical diagnosis. A gene cloned from chicken embryo fibroblast (CEF) cell was inserted into the expression vector pET-28a and pGEX-6p-1. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3). Then the recombinant proteins His-chGILZ and GST-chGILZ were successfully expressed and purified...
August 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28805498/gingiskhan%C3%A2-protease-cleavage-allows-a-high-throughput-antibody-to-fab-conversion-enabling-direct-functional-assessment-during-lead-identification-of-human-monoclonal-and-bispecific-igg1-antibodies
#3
Jörg Moelleken, Manuel Endesfelder, Christian Gassner, Sabine Lingke, Simone Tomaschek, Oksana Tyshchuk, Stefan Lorenz, Ulrike Reiff, Michael Mølhøj
The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs...
August 14, 2017: MAbs
https://www.readbyqxmd.com/read/28805373/inefficient-ribosomal-skipping-enables-simultaneous-secretion-and-display-of-proteins-in-saccharomyces-cerevisiae
#4
Carlos A Cruz-Teran, Karthik Tiruthani, Adam Mischler, Balaji M Rao
The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28805287/nongenetically-modified-lactococcus-lactis-adjuvanted-vaccination-enhanced-innate-immunity-against-helicobacter-pylori
#5
Wei Liu, Zhoulin Tan, Hai Liu, Zhiqin Zeng, Shuanghui Luo, Huimin Yang, Lufeng Zheng, Tao Xi, Yingying Xing
BACKGROUND: Gram-positive enhancer matrix particles (GEM) produced by Lactococcus lactis can enhance vaccine-induced immune response. However, the mechanism under which this adjuvant mounts the efficacy of orally administered vaccines remains unexplored. MATERIALS AND METHODS: We used a prophylactic mice model to investigate the mechanism of GEM-adjuvanted vaccination. Helicobacter pylori urease-specific antibody response was monitored and detected in murine serum by ELISA...
August 14, 2017: Helicobacter
https://www.readbyqxmd.com/read/28805269/cloning-and-bacterial-expression-systems-for-recombinant-human-heparanase-production-substrate-specificity-investigation-by-docking-of-a-putative-heparanase-substrate
#6
Angela Pennacchio, Alessandro Capo, Simonetta Caira, Annabella Tramice, Antonio Varriale, Maria Staiano, Sabato D'Auria
Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed from a subunit of 8 kDa and a subunit of 50 kDa. The two protein subunits are non-covalently associated. The cloning and expression of the two protein subunits in Escherichia coli, and their subsequent purification to homogeneity under native conditions result in the production of an active heparanase enzyme...
August 14, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28805010/characterization-of-hydrogels-made-of-a-novel-spider-silk-protein-emasp1s-and-evaluation-for-3d-printing
#7
Christopher Thamm, Elise DeSimone, Thomas Scheibel
Recombinantly produced spider silk proteins have high potential for bioengineering and various biomedical applications because of their biocompatibility, biodegradability, and low immunogenicity. Here, the recently described small spider silk protein eMaSp1s is assembled into hydrogels, which can be 3D printed into scaffolds. Further, blending with a recombinantly produced MaSp2 derivative eADF4(C16) alters the mechanical properties of the resulting hydrogels. Different spider silk hydrogels also show a distinct recovery after a high shear stress deformation, exhibiting the tunability of their features for selected applications...
August 14, 2017: Macromolecular Bioscience
https://www.readbyqxmd.com/read/28804955/3m-052-as-an-adjuvant-for-a-plga-microparticle-based-leishmania-donovani-recombinant-protein-vaccine
#8
Qian Wang, Meagan A Barry, Christopher A Seid, Elissa M Hudspeth, C Patrick McAtee, Michael J Heffernan
It is believed that an effective vaccine against leishmaniasis will require a T helper type 1 (TH 1) immune response. In this study, we investigated the adjuvanticity of the Toll-like receptor (TLR) 7/8 agonist 3M-052 in combination with the Leishmania donovani 36-kDa nucleoside hydrolase recombinant protein antigen (NH36). NH36 and 3M-052 were encapsulated in separate batches of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). The loading efficiency for NH36 was 83% and for 3M-052 was above 95%. In vitro stimulation of bone marrow-derived dendritic cells, measured by IL-12 secretion, demonstrated that 3M-052 (free or MP-formulated) had a concentration-dependent immunostimulatory effect with an optimum concentration of 2 µg/mL...
August 14, 2017: Journal of Biomedical Materials Research. Part B, Applied Biomaterials
https://www.readbyqxmd.com/read/28804866/virally-vectored-vaccine-delivery-medical-needs-mechanisms-advantages-and-challenges
#9
Daniel D Pinschewer
Vaccines represent one of the most successful chapters in the history of medicine. Over the past decades, the advent of recombinant cDNA technology has enabled the biomedical community to genetically engineer viruses for vaccine delivery purposes. As a starting point, this review evaluates the unmet medical needs, which drive scientists and industry to exploit such fundamentally new technology for human vaccination. The author discusses the molecular functioning, production and safety profile of replication-competent and -deficient viral vector systems, representing two fundamentally distinct classes of "genetic vaccines"...
August 14, 2017: Swiss Medical Weekly
https://www.readbyqxmd.com/read/28804843/diagnosis-or-exclusion-of-von-willebrand-disease-using-laboratory-testing
#10
Emmanuel J Favaloro
von Willebrand disease (VWD) is a common bleeding disorder diagnosed based on clinical features and following laboratory testing. VWD is due to deficiencies or defects in the plasma protein von Willebrand factor (VWF), a large adhesive protein with multiple activities. Laboratory testing therefore centers on assessment of VWF protein level using VWF antigen (VWF:Ag), as well as assays that measure VWF activity, most notably platelet glycoprotein (GP) Ib and collagen binding (VWF:CB) activities. Decreases in VWF:Ag and VWF activities, as well as the pattern of such changes, help define VWD and its type...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28804553/sirt3-attenuates-doxorubicin-induced-cardiac-hypertrophy-and-mitochondrial-dysfunction-via-suppression-of-bnip3
#11
Qiong Du, Bin Zhu, Qing Zhai, Bo Yu
Doxorubicin (Dox) is an anthracycline antibiotic widely used in cancer treatment. Although its antitumor efficacy appears to be dose dependent, its clinical use is greatly restricted by development of cardiotoxicity. Sirtuin-3 (Sirt3) is the major deacetylase within the mitochondrial matrix that plays an important role in regulation of cardiac function. This study was performed to identify the regulatory role of Sirt3 on Dox-induced cardiac hypertrophy and mitochondrial dysfunction in rats in vivo and in vitro...
2017: American Journal of Translational Research
https://www.readbyqxmd.com/read/28804448/erythropoietin-protects-against-lipopolysaccharide-induced-microgliosis-and-abnormal-granule-cell-development-in-the-ovine-fetal-cerebellum
#12
Annie R A McDougall, Nadia Hale, Sandra Rees, Richard Harding, Robert De Matteo, Stuart B Hooper, Mary Tolcos
Erythropoietin (EPO) ameliorates inflammation-induced injury in cerebral white matter (WM). However, effects of inflammation on the cerebellum and neuroprotective effects of EPO are unknown. Our aims were to determine: (i) whether lipopolysaccharide (LPS)-induced intrauterine inflammation causes injury to, and/or impairs development of the cerebellum; and (ii) whether recombinant human EPO (rhEPO) mitigates these changes. At 107 ± 1 days gestational age (DGA; ~0.7 of term), fetal sheep received LPS (~0.9 μg/kg; i...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28803330/bime2-a-novel-gene-required-for-interhomolog-meiotic-recombination-in-the-protist-model-organism-tetrahymena
#13
Anura Shodhan, Maria Novatchkova, Josef Loidl
Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Most DSBs are converted into nonreciprocal exchanges (gene conversions) or crossovers (COs) between sister chromatids. Only a minority of DSBs are processed toward interhomolog COs, the precursors of the chiasmata that connect homologous chromosomes. Dmc1, the meiosis-specific paralog of the universal recombination protein Rad51, is required for interhomolog COs; in its absence, univalents are primarily formed. Here, we report a ciliate-specific novel meiotic gene, BIME2, which also promotes interhomolog crossing over...
August 12, 2017: Chromosome Research
https://www.readbyqxmd.com/read/28803254/helicobacter-pylori-cytotoxin-associated-gene-a-impairs-the-filtration-barrier-function-of-podocytes-via-p38-mapk-signaling-pathway
#14
Man Yang, Ling Wang, Li-Jie Gu, Wei-Jie Yuan
Helicobacter pylori (Hp) specific antigens were found deposited in the glomeruli in some kidney diseases. However, the underlying molecular mechanisms remain to be elucidated. The aim of this study was to investigate the effect of cytotoxin associated gene A protein (CagA), a key virulence factor of Hp, on mouse podocytes. Cells were cultured and treated with recombinant CagA protein. The expression of the tight junction protein ZO-1 and p38 MAPK signaling pathway activation were measured with real-time RT-PCR and western blotting...
August 12, 2017: Acta Biochimica Polonica
https://www.readbyqxmd.com/read/28803053/optimized-transitory-ectopic-expression-of-promastigote-surface-antigen-protein-in-nicotiana-benthamiana-a-potential-anti-leishmaniasis-vaccine-candidate
#15
Séverine Lacombe, Martine Bangratz, Jean-Paul Brizard, Elodie Petitdidier, Julie Pagniez, Drissa Sérémé, Jean-Loup Lemesre, Christophe Brugidou
In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum...
August 10, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28802994/expression-and-purification-of-norovirus-virus-like-particles-in-escherichia-coli-and-their-immunogenicity-in-mice
#16
Yuqi Huo, Xin Wan, Tong Ling, Jie Wu, Wenhui Wang, Shuo Shen
Norovirus (NoV) virus like particles (VLPs) produced in Spodoptera frugiperda (Sf9) cells have been tested in human volunteers as vaccine candidate and were shown to be protective against NoV induced acute gastroenteritis. In this study, prevailing Sydney-2012-like NoV major capsid protein gene with or without N-terminal deletions (N26 and N38, 26 and 38 amino acids deleted from N terminus,respectively) were sub-cloned into prokaryotic expression vector, pCold III and pCold IV. Soluble and insoluble proteins were detected for both vectors after induction and higher levels of protein expression were observed for constructs pCold III-N26 and pCold III-N38...
August 9, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28802864/%C3%AE-3-adrenergic-receptor-regulates-hepatic-apolipoprotein-a-i-gene-expression
#17
Xiaojing Cao, Yanfang Li
BACKGROUND: β3-adrenergic receptor (β3-AR) was shown to upregulate hepatic apolipoprotein A-I (apoA-I) expression and reverse atherosclerotic plaques in vivo experiments. However, the effect of β3-AR on apoA-I expression in vitro is unknown. The specific mechanism underlying β3-AR prevention of atherosclerosis is unclear. OBJECTIVE: The present study was designed to investigate the molecular mechanism of β3-AR-mediated regulation of hepatic apoA-I gene expression...
July 27, 2017: Journal of Clinical Lipidology
https://www.readbyqxmd.com/read/28802644/rapid-characterization-of-the-cho-platform-cell-line-and-identification-of-pseudo-attp-sites-for-phic31-integrase
#18
Narges Damavandi, Mozhgan Raigani, Atefeh Joudaki, Fatemeh Davami, Sirous Zeinali
The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization...
August 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28802400/mycobacterium-tuberculosis-myma-is-a-tlr2-agonist-that-activate-macrophages-and-a-th1-response
#19
Iti Saraav, Swati Singh, Kirti Pandey, Monika Sharma, Sadhna Sharma
Cell wall of Mycobacterium tuberculosis (M.tb) is a major source of immunogenic proteins that can be tested as vaccine candidates. MymA (Rv3083), a 55 kDa M.tb flavin containing monooxygenase, is involved in modification of mycolic acids during acidic shock following M.tb internalization in macrophage. In this study, we have investigated the role of this cell wall associated protein in activation of macrophages by toll like receptor (TLRs) engagement and subsequent signaling. Our results showed that MymA stimulation of THP1 cells and human monocyte derived macrophages (MDM) lead to upregulation of TLR2 and co-stimulatory molecules CD40, CD80, CD86 and HLA-DR...
September 2017: Tuberculosis
https://www.readbyqxmd.com/read/28802265/syntrophomonas-wolfei-uses-a-nadh-dependent-ferredoxin-independent-fefe-hydrogenase-to-reoxidize-nadh
#20
Nathaniel A Losey, Florence Mus, John W Peters, Huynh M Le, Michael J McInerney
Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbon in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-CoA, formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were co-expressed in Escherichia coli and the recombinant Hyd1ABC was purified and characterized...
August 11, 2017: Applied and Environmental Microbiology
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