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https://www.readbyqxmd.com/read/28430399/targeting-conformational-activation-of-cdk2-kinase
#1
Morgan Pellerano, Sergey Tcherniuk, Corine Perals, Thi Nhu Ngoc Van, Elsa Garcin, Florence Mahuteau-Betzer, Marie-Paule Teulade-Fichou, May C Morris
Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation...
April 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28429856/glycosyltransferase-cascades-for-natural-product-glycosylation-use-of-plant-instead-of-bacterial-sucrose-synthases-improves-the-udp-glucose-recycling-from-sucrose-and-udp
#2
Alexander Gutmann, Alexander Lepak, Margo Diricks, Tom Desmet, Bernd Nidetzky
Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin...
April 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28429845/following-nature-s-roadmap-folding-factors-from-plasma-cells-led-to-improvements-in-antibody-secretion-in-s-cerevisiae
#3
Essi V Koskela, Jorg C de Ruijter, Alexander D Frey
Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite multiple numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, we demonstrate the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast...
April 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28418108/metabolic-characterization-and-transformation-of-the-non-dairy-lactococcus-lactis-strain-kf147-for-production-of-ethanol-from-xylose
#4
Kia Vest Petersen, Jianming Liu, Jun Chen, Jan Martinussen, Peter Ruhdal Jensen, Christian Solem
The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, we characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, we eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase...
April 18, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28403549/chaotropic-heat-treatment-resolves-native-like-aggregation-of-a-heterologously-produced-hyperthermostable-laminarinase
#5
Adrie H Westphal, Astrid A Geerke-Volmer, Carlo P M van Mierlo, Willem J H van Berkel
Production of hyperthermostable enzymes in mesophilic hosts frequently causes undesired aggregation of these proteins. During production of Pyrococcus furiosus endo-β-1,3 glucanase (LamA) in Escherichia coli, soluble and insoluble species form. Here, we address the composition of this mixture, including the nature of LamA conformers, and establish a method to increase the yield of native monomer. With gel electrophoresis, size-exclusion chromatography, light scattering, circular dichroism and enzyme kinetics we show that approximately 50 % of heterologously produced LamA is soluble, and that 40 % of this fraction constitutes native-like oligomers and non-native monomers...
April 12, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28398709/continuous-desalting-of-refolded-protein-solution-improves-capturing-in-ion-exchange-chromatography-a-seamless-process
#6
Nicole Walch, Alois Jungbauer
Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns...
April 11, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28397983/high-yield-production-of-recombinant-crm197-a-non-toxic-mutant-of-diphtheria-toxin-in-the-periplasm-of-escherichia-coli
#7
Philippe Goffina, Marianne Dewerchin, Philippe De Rop, Normand Blais, Philippe Dehottay
A high cell density fed-batch process was developed for production of recombinant CRM197, a non-toxic mutant of diphtheria toxin widely used as a carrier in polysaccharide-protein conjugate vaccines. Fully soluble recombinant CRM197 was obtained in high yields and with an authentic N-terminus, by targeting the protein to the periplasm of Escherichia coli using the Signal Recognition Particle (SRP)-dependent signal sequence of FlgI. Response Surface Methodology was used to optimize the set-points of key process parameters (pH and feed rate at induction)...
April 11, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371407/biocatalytic-virus-capsid-as-nanovehicle-for-enzymatic-activation-of-tamoxifen-in-tumor-cells
#8
Alejandro Tapia-Moreno, Karla Juarez-Moreno, Oscar Gonzalez-Davis, Ruben D Cadena-Nava, Rafael Vazquez-Duhalt
Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli...
April 3, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371347/genetic-engineering-of-the-branched-fatty-acid-metabolic-pathway-of-bacillus-subtilis-for-the-overproduction-of-surfactin-c14-isoform
#9
Debarun Dhali, François Coutte, Anthony Argüelles Arias, Sandrine Auger, Vladimir Bidnenko, Gabrielle Chataigné, Michael Lalk, Joachim Niehren, Joana de Sousa, Cristian Versari, Philippe Jacques
Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted...
April 3, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371300/a-single-step-facs-sorting-strategy-in-conjunction-with-fluorescent-vital-dye-imaging-efficiently-assures-clonality-of-biopharmaceutical-production-cell-lines
#10
Jürgen Fieder, Patrick Schulz, Ingo Gorr, Harald Bradl, Till Wenger
The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, we describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation...
April 3, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371185/a-fine-tuned-composition-of-protein-nanofibrils-yields-an-upgraded-functionality-of-displayed-antibody-binding-domains
#11
Benjamin Schmuck, Mats Sandgren, Torleif Härd
Elevated performance of instruments and electronic devices is frequently attained through miniaturization of the involved components, which increases the number of functional units in a given volume. Analogously, to conquer the limitations of materials used for the purification of monoclonal antibodies and for the sensitivity of immunoassays, the support for capturing antibodies requires miniaturization. A suitable scaffold for this purpose are cross-β structured protein nanofibrils, as they offer a superior surface area over volume ratio and because manipulation can be implemented genetically...
April 3, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371494/quality-by-control-towards-model-predictive-control-of-mammalian-cell-culture-bioprocesses
#12
REVIEW
Wolfgang Sommeregger, Bernhard Sissolak, Kulwant Kandra, Moritz von Stosch, Martin Mayer, Gerald Striedner
The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach...
March 31, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28371379/avian-embryos-and-related-cell-lines-a-convenient-platform-for-recombinant-proteins-and-vaccine-production
#13
REVIEW
Maryam Farzaneh, Seyedeh-Nafiseh Hassani, Paul Mozdziak, Hossein Baharvand
Chick embryos are a significant historical research model in basic and applied sciences. The embryonated eggs have been used for virus inoculation in order to vaccine production for nearly a century. Recently, avian eggs and cell lines derived from embryonated eggs have found wide application in biotechnology. This review will discuss about the unique characteristics of avian eggs in terms of safety, large scale and economical production of recombinant proteins. This system also provides the human-like glycosylation on target proteins and therefore can be considered as a suitable host for biomanufacturing of humanized monoclonal antibodies and therapeutic proteins...
March 29, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28345250/the-application-of-constitutively-solvent-tolerant-p-taiwanensis-vlb120%C3%AE-c%C3%AE-ttgv-for-stereospecific-epoxidation-of-toxic-styrene-alleviates-carrier-solvent-use
#14
Jan Volmer, Andreas Schmid, Bruno Bühler
For whole-cell biotransformations involving toxic organic compounds, two-liquid phase setups are typically applied employing an apolar extractive phase. Bis(2-ethylhexyl)phthalate (BEHP) has proven to be an ideal solvent for stereospecific styrene epoxidation with recombinant E. coli, providing excellent extractive properties and a high biocompatibility. In eco-efficiency evaluations, BEHP, however, has been identified as a critical factor regarding costs and environmental impact. In this study, the constitutive solvent tolerance of P...
March 27, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28301074/high-throughput-inclusion-body-sizing-nano-particle-tracking-analysis
#15
Wieland N Reichelt, Andreas Kaineder, Markus Brillmann, Lukas Neutsch, Alexander Taschauer, Hans Lohninger, Christoph Herwig
The expression of pharmaceutical relevant proteins in E. coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In literature, substantial effort has been invested into the quantification of IB size. But up to this date particle based methods to analyze physical IB properties of representative numbers of IBs lack sensitivity and/or lack orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells...
March 16, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28299901/dual-display-of-proteins-on-the-yeast-cell-surface-simplifies-quantification-of-binding-interactions-and-enzymatic-bioconjugation-reactions
#16
Sungwon Lim, Jeff E Glasgow, Maria Filsinger Interrante, Erica M Storm, Jennifer R Cochran
Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus...
March 16, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28296345/a-cleavable-self-assembling-tag-strategy-for-preparing-proteins-and-peptides-with-an-authentic-n-terminus
#17
Qing Zhao, Bihong Zhou, Xianxing Gao, Lei Xing, Xu Wang, Zhanglin Lin
Recombinant protein expression and purification remains a central need for biotechnology. Herein, we report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L6 KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored...
March 15, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28294579/experimental-validation-of-in-silico-estimated-biomass-yields-of-pseudomonas-putida-kt2440
#18
Sarah Beate Hintermayer, Dirk Weuster-Botz
Pseudomonas putida is rapidly becoming a microbial cell platform for biotechnological applications. In order to understand genotype-phenotype relationships genome scale models represent helpful tools. However, the validation of in silico predictions of genome scale models is a task that is rarely performed. In this study the theoretical biomass yields of Pseudomonas putida KT2440 were estimated for 57 different carbon sources based on a genome scale stoichiometric model applying flux balance analysis. The batch growth of P...
March 14, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28294544/identification-of-acetylcholinesterase-inhibitors-using-homogenous-cell-based-assays-in-quantitative-high-throughput-screening-platforms
#19
Shuaizhang Li, Ruili Huang, Samuel Solomon, Yitong Liu, Bin Zhao, Michael F Santillo, Menghang Xia
Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell-based assays in high-throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH-SY5Y)...
March 14, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28296355/biosensor-enabled-directed-evolution-to-improve-muconic-acid-production-in-saccharomyces-cerevisiae
#20
John M Leavitt, James M Wagner, Cuong Chi Tu, Alice Tong, Yanyi Liu, Hal S Alper
Muconic acid is a valuable platform chemical with potential applications in the production of polymers such as nylon and polyethylene terephthalate (PET). The conjugate base, muconate, was previously biosynthesized in the bacterial host Escherichia coli. Significant pathway engineering led to the first reported instance of rationally engineered production of muconic acid in the yeast Saccharomyces cerevisiae. To further increase muconic acid production in this host, we employed an adaptive laboratory evolution (ALE) strategy to complement rational metabolic engineering...
March 10, 2017: Biotechnology Journal
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