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Recombinant protein

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https://www.readbyqxmd.com/read/29777593/degradation-of-recombinant-proteins-by-cho-host-cell-proteases-is-prevented-by-matriptase-1-knock-out
#1
Holger Laux, Sandrine Romand, Sandro Nuciforo, Christopher J Farady, Joel Tapparel, Stine Buechmann-Moeller, Benjamin Sommer, Edward J Oakeley, Ursula Bodendorf
An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells...
May 19, 2018: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29777592/early-identification-of-unusually-clustered-mutations-and-a-root-cause-in-therapeutic-antibody-development
#2
Yueming Qian, Zhiqiang Chen, Xin Huang, Xuning Wang, Xuankuo Xu, Stefan Kirov, Richard Ludwig, Nan-Xin Qian, Kandasamy Ravi, Li Tao, Michael C Borys, Zheng Jian Li
This study reports findings of an unusual cluster of mutations spanning 22 base pairs in a monoclonal antibody (mAb) expression vector. It was identified by two orthogonal methods: mass spectrometry on expressed protein and next-generation sequencing (NGS) on the plasmid DNA. While the initial NGS analysis confirmed the designed sequence modification, intact mass analysis detected an additional mass of the antibody molecule expressed in CHO cells. The extra mass was eventually found to be associated with unmatched nucleotides in a distal region by checking full-length sequence alignment plots...
May 19, 2018: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29777551/a-new-method-for-quantitative-determination-of-renalase-based-on-mass-spectrometry-determination-of-a-proteotypic-peptide-labeled-with-stable-isotopes
#3
A T Kopylov, V I Fedchenko, O A Buneeva, N V Pyatakova, V G Zgoda, A E Medvedev
RATIONALE: Renalase is a recently discovered kidney secretory protein, which is considered as an important component involved in blood pressure regulation. Although altered levels of renalase have been detected in plasma and urine of patients with various kidney diseases, there is certain inconsistency of changes in the renalase levels reported by different laboratories. The latter is obviously associated with the use of ELISA as the only available approach for quantitative analysis of renalase...
May 18, 2018: Rapid Communications in Mass Spectrometry: RCM
https://www.readbyqxmd.com/read/29777512/discovery-of-an-acidic-thermostable-and-highly-nadp-dependent-formate-dehydrogenase-from-lactobacillus-buchneri-nrrl-b-30929
#4
Saadet Alpdağtaş, Sevil Yücel, Handan Açelya Kapkaç, Siqing Liu, Barış Binay
OBJECTIVES: To identify a robust NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. RESULTS: A new NADP+ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 × histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE...
May 18, 2018: Biotechnology Letters
https://www.readbyqxmd.com/read/29777363/high-level-production-of-single-chain-monellin-mutants-with-enhanced-sweetness-and-stability-in-tobacco-chloroplasts
#5
Daniela Castiglia, Serena Leone, Rachele Tamburino, Lorenza Sannino, Jole Fonderico, Chiara Melchiorre, Andrea Carpentieri, Stefania Grillo, Delia Picone, Nunzia Scotti
Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins. The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications...
May 18, 2018: Planta
https://www.readbyqxmd.com/read/29777164/mrna-treatment-produces-sustained-expression-of-enzymatically-active-human-adamts13-in-mice
#6
Susan Liu-Chen, Brendan Connolly, Lei Cheng, Romesh R Subramanian, Zhaozhong Han
Thrombotic thrombocytopenic purpura (TTP) is primarily caused by deficiency of ADAMTS13 within the blood stream due to either genetic defects or presence of inhibitory autoantibodies. Preclinical and clinical studies suggest that enzyme replacement therapy with recombinant human ADAMTS13 protein (rhADAMTS13) is effective and safe in treatment of TTP. However, frequent dosing would be required due to the relatively short half-life of rhADAMTS13 in circulation as well as the presence of inhibitory autoantibodies that collectively result in the poor pharmacological profile of rhADAMTS13...
May 18, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29777059/sequence-diversity-of-tubulin-isotypes-in-regulation-of-the-mitochondrial-voltage-dependent-anion-channel
#7
Tatiana K Rostovtseva, Philip A Gurnev, David P Hoogerheide, Amandine Rovini, Minhajuddin Sirajuddin, Sergey M Bezrukov
The microtubule protein tubulin is a heterodimer comprising α/β subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight β-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic C-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin's role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC)...
May 18, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29776931/shuffling-the-neutral-drift-of-unspecific-peroxygenase-in-saccharomyces-cerevisiae
#8
Javier Martin-Diaz, Carmen Paret, Eva García-Ruiz, Patricia Molina-Espeja, Miguel Alcalde
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded and active UPO variants were generated...
May 18, 2018: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29776374/the-toxoplasma-gondii-me-49-strain-upregulates-levels-of-a20-that-inhibit-nf-%C3%AE%C2%BAb-activation-and-promotes-apoptosis-in-human-leukaemia-t-cell-lines
#9
Qian Chen, Min-Hui Pang, Xiao-Hong Ye, Guang Yang, Chen Lin
BACKGROUND: Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear...
May 18, 2018: Parasites & Vectors
https://www.readbyqxmd.com/read/29775888/therapeutic-impact-of-rhuepo-on-abnormal-platelet-app-bace-1-presenilin-1-adam-10-and-a%C3%AE-expressions-in-chronic-kidney-disease-patients-with-cognitive-dysfunction-like-alzheimer-s-disease-a-pilot-study
#10
Vinothkumar G, Krishnakumar S, Sureshkumar, Shivashekar G, Sreedhar S, Preethikrishnan, Dinesh S, Sundaram A, Balakrishnan D, Riya, Venkataraman P
BACKGROUND: Cognitive dysfunction is reported to be a major cause of morbidity in chronic kidney disease (CKD). The senile plaques (SPs) in the brain are one of the most pathophysiological characteristics of cognitive dysfunction and its major constituent amyloid β (Aβ) released from amyloid precursor protein (APP) by β (BACE1) and γ (presenilin 1) secretases . Platelets contain more than 95% of the circulating APP and implicate as a candidate biomarker for cognitive decline. Recombinant human erythropoietin (rHuEPO) is a standard therapy for anemia in CKD and also acts as a neuroprotective agent...
May 14, 2018: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
https://www.readbyqxmd.com/read/29775866/identification-of-udp-glucosyltransferases-from-the-aluminum-resistant-tree-eucalyptus-camaldulensis-forming-%C3%AE-glucogallin-the-precursor-of-hydrolyzable-tannins
#11
Ko Tahara, Mitsuru Nishiguchi, Andrej Frolov, Juliane Mittasch, Carsten Milkowski
In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-β-d-glucose (β-glucogallin), the precursor of hydrolyzable tannins...
May 15, 2018: Phytochemistry
https://www.readbyqxmd.com/read/29775758/lncrna-prncr1-regulates-osteogenic-differentiation-in-osteolysis-after-hip-replacement-by-targeting-mir-211-5p
#12
Zong-Ming Gong, Zhen-Yu Tang, Xiao-Liang Sun
BACKGROUND: Osteogenic differentiation and osteolysis after hip replacement are both associated with bone metabolism. Interaction between lncRNA PRNCR1 and miR-211-5p was analyzed to illuminate their roles in osteogenic differentiation and osteolysis. METHODS: The expression of PRNCR1, miR-211-5p and C-X-C chemokine receptor-4 (CXCR4) protein in tissues and mesenchymal stem cells (MSCs) were determined by qRT-PCR and Western blot, separately. The osteogenic differentiation was assessed by ALP activity detection and ARS staining...
May 15, 2018: Gene
https://www.readbyqxmd.com/read/29775739/effects-of-environmental-factors-on-c-type-lectin-recognition-to-zooxanthellae-in-the-stony-coral-pocillopora-damicornis
#13
Zhi Zhou, Shuimiao Zhao, Junyi Ni, Yilu Su, Lingui Wang, Yanlai Xu
C-type lectin is a superfamily of Ca2+ -dependent carbohydrate-recognition proteins that play significant roles in nonself-recognition and pathogen clearance. In the present study, a C-type lectin (PdC-Lectin) was chosen from stony coral Pocillopora damicornis to understand its recognition characteristics to zooxanthellae. PdC-Lectin protein contained a signal peptide and a carbohydrate-recognition domain with EPN motif in Ca2+ -binding site 2. The PdC-Lectin recombinant protein was expressed and purified in vitro...
May 15, 2018: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/29775664/auto-induction-for-high-level-production-of-biologically-active-reteplase-in-escherichia-coli
#14
Mehrnoosh Fathi-Roudsari, Nader Maghsoudi, Amirhossein Maghsoudi, Sepideh Niazi, Morvarid Soleiman
Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E...
May 15, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29775652/advances-and-prospects-of-bacillus-subtilis-cellular-factories-from-rational-design-to-industrial-applications
#15
REVIEW
Yang Gu, Xianhao Xu, Yaokang Wu, Tengfei Niu, Yanfeng Liu, Jianghua Li, Guocheng Du, Long Liu
Bacillus subtilis is the most characterized gram-positive bacterium that has significant attributes, such as growing well on cheap carbon sources, possessing clear inherited backgrounds, having mature genetic manipulation methods, and exhibiting robustness in large-scale fermentations. Till date, B. subtilis has been identified as attractive hosts for the production of recombinant proteins and chemicals. By applying various systems and synthetic biology tools, the productivity features of B. subtilis can be thoroughly analyzed and further optimized via metabolic engineering...
May 15, 2018: Metabolic Engineering
https://www.readbyqxmd.com/read/29775464/biochemical-characterization-of-recbcd-enzyme-from-an-antarctic-pseudomonas-species-and-identification-of-its-cognate-chi-%C3%AF-sequence
#16
Theetha L Pavankumar, Anurag K Sinha, Malay K Ray
Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P...
2018: PloS One
https://www.readbyqxmd.com/read/29774745/interplay-between-copper-neprilysin-and-n-truncation-of-%C3%AE-amyloid
#17
Mariusz Mital, Wojciech Bal, Tomasz Frączyk, Simon C Drew
Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+ -dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition...
May 18, 2018: Inorganic Chemistry
https://www.readbyqxmd.com/read/29774653/matrix-glycoprotein-interactions-required-for-budding-of-a-plant-nucleorhabdovirus-and-induction-of-inner-nuclear-membrane-invagination
#18
Kai Sun, Xin Zhou, Wenye Lin, Xueping Zhou, Andrew O Jackson, Zhenghe Li
Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein-nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino-terminal (NT) or carboxyl-terminal (CT) domain...
May 18, 2018: Molecular Plant Pathology
https://www.readbyqxmd.com/read/29774570/high-mobility-group-box-1-drives-fibrosis-progression-signaling-via-the-receptor-for-advanced-glycation-end-products-in-mice
#19
Xiaodong Ge, Elena Arriazu, Fernando Magdaleno, Daniel J Antoine, Rouchelle Dela Cruz, Neil Theise, Natalia Nieto
BACKGROUND & RATIONALE: High-mobility group box-1 (HMGB1) is a damage-associated molecular pattern (DAMP) increased in response to liver injury. Since HMGB1 is a ligand for the receptor for advanced glycation end-products (RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis via RAGE cell-specific signaling mechanisms. RESULTS: liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic Hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC) and alcoholic steatohepatitis (ASH)...
May 18, 2018: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29774544/functional-molecules-in-mesothelial-to-mesenchymal-transition-revealed-by-transcriptome-analyses
#20
Sara Namvar, Adrian S Woolf, Leo A H Zeef, Thomas Wilm, Bettina Wilm, Sarah E Herrick
Peritoneal fibrosis is a common complication of abdominal and pelvic surgery, and can also be triggered by peritoneal dialysis, resulting in treatment failure. In these settings, fibrosis is driven by activated myofibroblasts that are considered to be partly derived by mesothelial-to-mesenchymal transition (MMT). We hypothesised that if the molecular signature of MMT could be better defined, these insights could be exploited to block this pathological cellular transition. Rat peritoneal mesothelial cells were purified, using an antibody to HBME1, a protein present on mesothelial cell microvilli, and streptavidin nanobead technology...
May 17, 2018: Journal of Pathology
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