Read by QxMD icon Read

protein purification

Cristian De Gregorio, Paula Díaz, Rodrigo López-Leal, Patricio Manque, Felipe A Court
Exosomes are small (30-150 nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear...
2018: Methods in Molecular Biology
Peggy Stolt-Bergner, Christian Benda, Tim Bergbrede, Hüseyin Besir, Patrick H N Celie, Cindy Chang, David Drechsel, Ariane Fischer, Arie Geerlof, Barbara Giabbai, Joop van den Heuvel, Georg Huber, Wolfgang Knecht, Anita Lehner, Regis Lemaitre, Kristina Nordén, Gwynn Pardee, Ines Racke, Kim Remans, Astrid Sander, Judith Scholz, Magda Stadnik, Paola Storici, Daniel Weinbruch, Isabel Zaror, Linda H L Lua, Sabine Suppmann
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E...
March 12, 2018: Journal of Structural Biology
V Vasilca, A Sadeghpour, S Rawson, L E Hawke, S A Baldwin, T Wilkinson, D Bannister, V L G Postis, M Rappolt, S P Muench, L J C Jeuken
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment...
March 12, 2018: Analytical Biochemistry
Alborz Karimzadeh, Vanessa M Scarfone, Erika Varady, Connie Chao, Karin Grathwohl, John W Fathman, David A Fruman, Thomas Serwold, Matthew A Inlay
Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs...
March 15, 2018: Stem Cells Translational Medicine
Ingo de Vries, Sarah Schreiber, Daniel Boßmann, Zawadi Hellmann, Jens Kopatz, Harald Neumann, Sascha Beutel
Polysialic acid (polySia) is a promising molecule for various medical applications (e.g., treatment of inflammatory neurodegenerative diseases). In this study a complete production process for human-identical α-(2,8)-linked polySia was developed using a disposable bioreactor for cultivation of Escherichia coli K1 and single-use membrane adsorbers for downstream processing (DSP). The cultivation process was optimized to minimize complex media components and a maturation process after cultivation was established...
March 2018: Biotechnology Reports
Sheun Oshinbolu, Rachana Shah, Gary Finka, Mike Molloy, Mark Uden, Daniel G Bracewell
BACKGROUND: A current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis-ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures. RESULTS: The null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis-ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%...
March 2018: Journal of Chemical Technology and Biotechnology
Héctor Miranda-Astudillo, Lilia Colina-Tenorio, Alejandra Jiménez-Suárez, Miriam Vázquez-Acevedo, Bénédicte Salin, Marie-France Giraud, Claire Remacle, Pierre Cardol, Diego González-Halphen
The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decade's most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F1 FO -ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V2 and V4 ) and different respiratory supercomplexes (I/IV6 , I/III4 , I/IV)...
March 11, 2018: Biochimica et Biophysica Acta
Sureyya Mert Selimoglu, Murat Kasap, Gurler Akpinar, Aynur Karadenizli, Abdul Mounem Wis, Uzay Gormus
Human creatine kinase MB (hCKMB) is one of the most preferred biomarkers used for the diagnosis of acute coronary syndrome due to its high sensitivity and specificity. The increasing need for highly purified and biologically active hCKMB in the field of diagnostics makes its production valuable. Currently, the production of hCKMB is mainly achieved in methylotrophic yeast, Pichia pastoris, because the production in Escherichia coli is challenging and generally yields an inactive enzyme with a low quantity. With the aim of finding the best way for the high-yield production of active hCKMB in E...
March 14, 2018: Journal of Molecular Microbiology and Biotechnology
Mahboubeh Kheirabadi, Gardner S Creech, Jennifer X Qiao, David S Nirschl, David K Leahy, Kenneth M Boy, Percy H Carter, Martin D Eastgate
Macrocyclic peptides containing N-alkylated amino acids have emerged as a promising therapeutic modality, capable of modulating protein-protein interactions and intracellular delivery of hydrophilic payloads. While multi-channel automated Solid Phase Peptide Synthesis (SPPS) is a practical approach for peptide synthesis, the requirement for slow and inefficient chromatographic purification of the product peptides is a significant limitation to exploring these novel compounds. Herein, we invent a "catch-release" strategy for the non-chromatographic purification of macrocyclic peptides...
March 14, 2018: Journal of Organic Chemistry
James Gilbert Atherton, David S Hains, John J Bissler, Bradford D Pendley, Erno Lindner
Current dialysis dosing calculations provide an incomplete assessment of blood purification. They do not include clearances of protein-bound uremic toxins like polyamines, p-cresol sulfate, and indoxyl sulfate, relying solely on the clearance of urea as a surrogate for all molecules accumulating in patients with end stage renal disease (ESRD). Protein-bound uremic toxins clear differently in dialysis but also during normal renal function. The kidney clears protein bound toxins via the process of secretion, while it clears urea through filtration...
March 14, 2018: American Journal of Physiology. Renal Physiology
Kelvin J Yong, Tasneem M Vaid, Patrick J Shilling, Feng-Jie Wu, Lisa M Williams, Mattia Deluigi, Andreas Plückthun, Ross Ad Bathgate, Paul R Gooley, Daniel J Scott
α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene super-family is made up of numerous sub-families that, like α1A-AR and α1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads...
March 14, 2018: ACS Chemical Biology
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC...
2018: Journal of Extracellular Vesicles
Imran Khan, Patricia S Steeg
The NM23/NME gene was identified as a metastasis suppressor. It's re-expression inhibited cancer cell motility and suppressed metastasis, without effecting primary tumor size in multiple model systems. The mechanisms of NME suppression of motility and metastasis are incompletely known. Of particular interest, has been NME histidine 118 phosphorylation, involved in nucleoside diphosphate kinase (NDPK) and histidine protein kinase (HPK) activities. Using recently developed monoclonal antibodies to phosphohistidine, we have addressed the correlation of NME phosphohistidine with motility suppression, and distinguished the NDPK and HPK contributions...
February 13, 2018: Oncotarget
Chady Nasrallah, Karine Rottier, Romain Marcellin, Vincent Compan, Joan Font, Amadeu Llebaria, Jean-Philippe Pin, Jean-Louis Banères, Guillaume Lebon
The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism...
March 13, 2018: Scientific Reports
Tomoyuki Hatano, Salvatore Alioto, Emanuele Roscioli, Saravanan Palani, Scott T Clarke, Anton Kamnev, Juan Ramon Hernandez-Fernaud, Lavanya Sivashanmugam, Bernardo Chapa-Y-Lazo, Alexandra M E Jones, Robert C Robinson, Karuna Sampath, Masanori Mishima, Andrew D McAinsh, Bruce L Goode, Mohan K Balasubramanian
Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion...
March 13, 2018: Journal of Cell Science
N Wang, Q Xu, Y Liu, Y Jin, P W Harlina, M Ma
Low-density lipoprotein (LDL) from hen egg yolk has high nutritional value and plays an important role in the fields of biology, medicine, and materials. To develop fundamental research about LDL, a highly efficient extraction method is necessary. We found that 30% saturated ammonium sulfate can extract more crude LDL than 40% saturation. We selected polyethylene glycol (PEG; nonionic type) to obtain crude LDL. Three factors were employed, namely, degree of polymerization, concentration of PEG, and pH of egg yolk plasma...
March 8, 2018: Poultry Science
Stanley Chung, Jun Tian, Zhijun Tan, Jie Chen, Jongchan Lee, Michael Borys, Zheng Jian Li
Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts towards developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants...
March 13, 2018: Biotechnology and Bioengineering
Jun Jiang, Shulin Tang, Jianhong Xia, Jikai Wen, Shuang Chen, Xiaodong Shu, Michael S Y Huen, Yiqun Deng
Wnt/β-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants. However, the molecular components and the regulatory mechanisms involved in its fine-tuning remain to be determined. Here, we identified C9orf140, a tumor-specific protein, as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry. We further showed that C9orf140 is a negative regulator of Wnt/β-catenin signaling in cultured cells as well as in zebrafish embryos. It functions upstream of β-catenin, outcompetes PP2A for binding to Axin1, influences the balance between phosphorylation and de-phosphorylation of β-catenin, and ultimately compromises Wnt3A-induced β-catenin accumulation...
March 13, 2018: Oncogene
Jeffrey W Habig, Aaron Rowland, Matthew G Pence, Cathy X Zhong
Resistance genes (R-genes) from wild potato species confer protection against disease and can be introduced into cultivated potato varieties using breeding or biotechnology. The R-gene, Rpi-vnt1, which encodes the VNT1 protein, protects against late blight, caused by Phytophthora infestans. Heterologous expression and purification of active VNT1 in quantities sufficient for regulatory biosafety studies was problematic, making it impractical to generate hazard characterization data. As a case study for R-proteins, a weight-of-evidence, tiered approach was used to evaluate the safety of VNT1...
March 9, 2018: Regulatory Toxicology and Pharmacology: RTP
E Quintero-Troconis, N Buelvas, C Carrasco-López, M R Domingo-Sananes, L González-González, R Ramírez-Molina, L Osorio, A Lobo-Rojas, A J Cáceres, P A Michels, H Acosta, W Quiñones, J L Concepción
Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain...
March 9, 2018: Biochimica et Biophysica Acta
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"