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https://www.readbyqxmd.com/read/29150912/sustaining-an-efficient-and-effective-cho-cell-line-development-platform-by-incorporation-of-24-deep-well-plate-screening-and-multivariate-analysis
#1
Alessandro Mora, Sheng Sam Zhang, Gerald Carson, Bernard Nabiswa, Patrick Hossler, Seongkyu Yoon
Efficient and effective cell line screening is paramount towards a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in 24-deep well plates (24-DWP) and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody...
November 18, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/29137641/improving-e-coli-growth-performance-by-manipulating-small-rna-expression
#2
REVIEW
Alejandro Negrete, Joseph Shiloach
Efficient growth of E. coli, especially for production of recombinant proteins, has been a challenge for the biotechnological industry since the early 1970s. By employing multiple approaches, such as different media composition, various growth strategies and specific genetic manipulations, it is now possible to grow bacteria to concentrations exceeding 100 g/L and to achieve high concentrations of recombinant proteins. Although the growth conditions are carefully monitored and maintained, it is likely that during the growth process cells are exposed to periodic stress conditions, created by fluctuations in pH, dissolved oxygen, temperature, glucose, and salt concentration...
November 14, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29113894/combined-effect-of-gene-dosage-and-process-optimization-strategies-on-high-level-production-of-recombinant-human-interleukin-3-hil-3-in-pichia-pastoris-fed-batch-culture
#3
Vikas Kumar Dagar, Yogender Pal Khasa
In this work, the combined effects of gene dosage and process optimization strategies were studied to achieve higher hIL-3 expression in Pichia system. The in-vitro multimerization method was used to generate various Pichia X-33 transformants having multi-copy expression cassettes. The quantitative polymerase chain reaction (qPCR) strategy was used to further confirm the genome integration of hIL-3 expression cassette. From shake flask expression studies, the recombinant hIL-3 concentration in culture supernatant increased upto 8 copies to a level of 310mg/L, thereafter a considerably lower expression was observed...
November 4, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29110014/tmao-mediates-effective-attraction-between-lipid-membranes-by-partitioning-unevenly-between-bulk-and-lipid-domains
#4
Shahar Sukenik, Shaked Dunsky, Avishai Barnoy, Ilan Shumilin, Daniel Harries
Under environmental duress, many organisms accumulate large amounts of osmolytes - molecularly small organic solutes. Osmolytes are known to counteract stress, driving proteins to their compact native states by their exclusion from protein surfaces. In contrast, the effect of osmolytes on lipid membranes is poorly understood and widely debated. Many fully membrane-permeable osmolytes exert an apparent attractive force between lipid membranes, yet all proposed models fail to fully account for the origin of this force...
November 7, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29078278/overcoming-factors-limiting-high-solids-fermentation-of-lignocellulosic-biomass-to-ethanol
#5
Thanh Yen Nguyen, Charles M Cai, Rajeev Kumar, Charles E Wyman
Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29040199/in-vitro-effect-of-lysozyme-on-albumin-deposition-to-hydrogel-contact-lens-materials
#6
Negar Babaei Omali, Lakshman N Subbaraman, Miriam Heynen, Zohra Fadli, Chantal Coles-Brennan, Lyndon W Jones
SIGNIFICANCE: Albumin deposition on contact lenses could be detrimental to contact lens (CL) wear because this may increase the risk of bacterial binding and reduce comfort. Lysozyme deposition on selected lens materials would reduce albumin deposition on lenses. PURPOSE: This study aims to determine if lysozyme deposition on CLs could act as a barrier against subsequent albumin adsorption, using an in vitro model. METHODS: Six hydrogel CL materials (etafilcon A, polymacon, nelfilcon A, omafilcon A, ocufilcon B, and nesofilcon A) were evaluated...
November 2017: Optometry and Vision Science: Official Publication of the American Academy of Optometry
https://www.readbyqxmd.com/read/29035529/md-and-qm-mm-study-of-the-quaternary-hutp-homohexamer-complex-with-mrna-l-histidine-ligand-and-mg-2
#7
Pavlína Pokorná, Miroslav Krepl, Holger Kruse, Jiří Šponer
The HutP protein from B. subtilis regulates histidine metabolism by interacting with an antiterminator mRNA hairpin in response to the binding of l-histidine and Mg(2+). We studied the functional ligand-bound HutP hexamer complexed with two mRNAs using all-atom microsecond-scale explicit-solvent MD simulations performed with the Amber force fields. The experimentally observed protein-RNA interface exhibited good structural stability in the simulations with the exception of some fluctuations in an unusual adenine-threonine interaction involving two closely spaced H-bonds...
November 1, 2017: Journal of Chemical Theory and Computation
https://www.readbyqxmd.com/read/29027448/-gene-optimization-and-efficient-expression-of-trichoderma-reesei-cel5a-in-pichia-pastoris
#8
Renhui Bai, Yunbo Zhang, Chundi Wang, Feiyang Zhang, Zhe Zhang, Fubao Sun, Zhenyu Zhang
Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28971699/cgrp-receptor-antagonist-mk-8825-attenuates-cortical-spreading-depression-induced-pain-behavior
#9
Aslı Filiz, Nermin Tepe, Sajedeh Eftekhari, H Evren Boran, Ergin Dilekoz, Lars Edvinsson, Hayrunnisa Bolay
Background and objective The present study aimed to investigate the effects of selective calcitonin gene related peptide (CGRP) receptor antagonist (MK-8825) on cortical spreading depression (CSD) induced pain behavior and anxiety in freely-moving rats, and neuronal activation in the correlated anatomical regions. Methods CSD was induced while keeping all meningeal layers and BBB intact and MK-8825 was administered in two different doses. Regional cerebral blood flow (rCBF), arterial pressure and DC shift were recorded...
January 1, 2017: Cephalalgia: An International Journal of Headache
https://www.readbyqxmd.com/read/28945068/microwave-assisted-protein-digestion-in-a-plate-well-for-facile-sampling-and-rapid-digestion
#10
Hyeonil Kim, Han Sol Kim, Dabin Lee, Dongwon Shin, Daeho Shin, Jeongkwon Kim, Jungbae Kim
Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwave-assisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively...
September 25, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28902870/reduced%C3%A2-insulin-signaling-maintains-electrical-transmission-in-a-neural-circuit-in-aging-flies
#11
Hrvoje Augustin, Kieran McGourty, Marcus J Allen, Sirisha Kudumala Madem, Jennifer Adcott, Fiona Kerr, Chi Tung Wong, Alec Vincent, Tanja Godenschwege, Emmanuel Boucrot, Linda Partridge
Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the "insulin paradox"). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced...
September 2017: PLoS Biology
https://www.readbyqxmd.com/read/28898422/dynamic-gastric-digestion-of-a-commercial-whey-protein-concentrate
#12
Beatriz Miralles, Roberto Del Barrio, Carolina Cueva, Isidra Recio, Lourdes Amigo
BACKGROUND: A dynamic gastrointestinal simulator, simgi(®) , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. RESULTS: Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively...
September 12, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28887386/fragile-x-mental-retardation-protein-restricts-small-dye-iontophoresis-entry-into-central-neurons
#13
Tyler Kennedy, Kendal Broadie
Fragile X mental retardation protein (FMRP) loss causes Fragile X syndrome (FXS), a major disorder characterized by autism, intellectual disability, hyperactivity, and seizures. FMRP is both an RNA- and channel-binding regulator, with critical roles in neural circuit formation and function. However, it remains unclear how these FMRP activities relate to each other and how dysfunction in their absence underlies FXS neurological symptoms. In testing circuit level defects in the Drosophila FXS model, we discovered a completely unexpected and highly robust neuronal dye iontophoresis phenotype in the well mapped giant fiber (GF) circuit...
October 11, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/28884428/in-vitro-and-in-vivo-anti-proliferative-evaluation-of-bis-4-4-tolyl-2-2-6-2%C3%A2-terpyridine-copper-ii-complex-against-ehrlich-ascites-carcinoma-tumors
#14
Dharmasivam Mahendiran, Raju Senthil Kumar, Vijayan Viswanathan, Devadasan Velmurugan, Aziz Kalilur Rahiman
The bis(4'-(4-tolyl)-2,2':6',2″-terpyridine)copper(II) complex [Cu(ttpy)2]Cl2 was synthesized and authenticated by single crystal analysis, which shows distorted octahedral geometry around copper(II) ion. The crystal packing is stabilized by C-H···π inter and intramolecular interactions. The complex was found to be lipophilic as determined by shake-flask method. In vitro cytotoxicity of the complex was tested against Ehrlich ascites carcinoma (EAC) and L6 myotube cell lines. The complex exhibit potent cytotoxicity with respect to the commercially available anticancer drug cisplatin...
October 2017: Journal of Biological Inorganic Chemistry: JBIC
https://www.readbyqxmd.com/read/28873703/oil-in-water-emulsions-stabilized-by-tyrosinase-crosslinked-potato-protein
#15
Jovana Glusac, Sivan Isaschar-Ovdat, Biljana Kukavica, Ayelet Fishman
Potato protein (PP) holds great promise as a non-allergenic food ingredient with high nutritional value. Attempts to modulate its functional properties by crosslinking have not been reported to date. The effect of tyrosinase-mediated crosslinking of PP on the properties of o/w emulsions was studied in the present work. Among the various PPs, protease inhibitors were efficiently crosslinked by the enzyme as determined by SDS-PAGE analysis. Concentrated emulsions comprising 40% olive oil and 6.1% PP (w/w) were fabricated by a shear stress homogenizer...
October 2017: Food Research International
https://www.readbyqxmd.com/read/28869737/-a-method-to-increase-the-production-of-insulin-precursor-in-pichia-pastoris
#16
Chenchen Liang, Li Wang, Qiuling Luo, Jing Wu
To improve the yield of insulin precursor (PI), we constructed a recombinant expression vector pPIC9K-PI and transformed it into Pichia pastoris GS115 using electroporation. After screening, a mutant strain CL012 with 12 copies was obtained on the YPDS plate containing 4.0 mg/mL G418. Then, the components of SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), SNC2 and SNC2-SSO2, were expressed in the strain CL012 to explore the effect of SNAREs on the yield of PI. In shake flask culture, the strains expressing SNC2 and SNC2-SSO2 yielded PI of 1...
July 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28865117/development-of-a-general-defined-medium-for-pichia-pastoris
#17
Catherine B Matthews, Angel Kuo, Kerry R Love, J Christopher Love
Pichia pastoris is widely used as a host for recombinant protein production. More than 500 proteins have been expressed in the organism at a variety of cultivation scales, from small shake flasks to large bioreactors. Large-scale fermentation strategies typically employ chemically defined growth medium because of its greater batch-to-batch consistency and in many cases, lower costs compared to complex medium. For biopharmaceuticals, defined growth medium may also simplify downstream purification and regulatory documentation...
September 2, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28861662/extracellular-expression-in-aspergillus-niger-of-an-antibody-fused-to-leishmania-sp-antigens
#18
Denis Magaña-Ortíz, Francisco Fernández, Achim M Loske, Miguel A Gómez-Lim
Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier...
August 31, 2017: Current Microbiology
https://www.readbyqxmd.com/read/28861059/characterization-of-full-length-and-truncated-recombinant-%C3%AE%C2%BA-carrageenase-expressed-in-pichia-pastoris
#19
Yuan Yu, Zhemin Liu, Min Yang, Meng Chen, Zhihan Wei, Lixia Shi, Li Li, Haijin Mou
κ-Carrageenase belongs to glycoside hydrolase family 16 and cleaves the β-(1→4) linkages of κ-carrageenan. In this study, genes encoding the full-length (cgkZ), Por secretion tail-truncated (cgkZΔPst) and carbohydrate binding domain-truncated (cgkZΔCBM) κ-carrageenase proteins were expressed in Pichia pastoris. The copy numbers of gene cgkZ, cgkZΔPst and cgkZΔCBM were 7, 7 and 6, respectively. The enzymatic activities of recombinant enzymes cgkZ, cgkZΔPst and cgkZΔCBM reached 4.68, 5.70, and 3.02 U/mL, respectively, after 120 h of shake flask fermentation at 22°C and pH 6 in the presence of 1 % (v/v) methanol...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28860027/preparation-of-wool-follicles-for-proteomic-studies
#20
Jeffrey E Plowman, Joy L Woods, Bede van Schaijik, Duane P Harland
A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs...
August 30, 2017: Analytical Biochemistry
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