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https://www.readbyqxmd.com/read/28088451/efficient-sortase-mediated-n-terminal-labeling-of-tev-protease-cleaved-recombinant-proteins
#1
Kwabena Sarpong, Ron Bose
A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein...
January 11, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28088197/production-of-human-pro-relaxin-h2-in-the-yeast-pichia-pastoris
#2
D Cimini, K Della Corte, R Finamore, L Andreozzi, A Stellavato, A V A Pirozzi, F Ferrara, R Formisano, M De Rosa, M Chino, L Lista, A Lombardi, V Pavone, C Schiraldi
BACKGROUND: Initially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production...
January 14, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28087714/dlg5-connects-cell-polarity-and-hippo-signaling-protein-networks-by-linking-par-1-with-mst1-2
#3
Julian Kwan, Anna Sczaniecka, Emad Heidary Arash, Liem Nguyen, Chia-Chun Chen, Srdjana Ratkovic, Olga Klezovitch, Liliana Attisano, Helen McNeill, Andrew Emili, Valeri Vasioukhin
Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation...
December 15, 2016: Genes & Development
https://www.readbyqxmd.com/read/28087367/production-of-recombinant-proteins-in-escherichia-coli-tagged-with-the-fusion-protein-cusf3h
#4
Teresa Vargas-Cortez, Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria, Xristo Zarate
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E...
January 10, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28078416/poly-ethylene-glycol-functionalization-of-monolithic-poly-divinyl-benzene-for-improved-miniaturized-solid-phase-extraction-of-protein-rich-samples
#5
Esme Candish, Aminreza Khodabandeh, Marianne Gaborieau, Thomas Rodemann, Robert A Shellie, Andrew A Gooley, Emily F Hilder
Non-specific protein adsorption on hydrophobic solid phase extraction (SPE) adsorbents can reduce the efficacy of purification. To improve sample clean-up, poly(divinyl benzene) (PDVB) monoliths grafted with hydrophilic polyethylene glycol methacrylate (PEGMA) were developed. Residual vinyl groups (RVGs) of the PDVB were employed as anchor points for PEGMA grafting. Two PEGMA monomers, M n 360 and 950, were compared for graft solutions containing 5-20% monomer. Protein binding was qualitatively screened using fluorescently labeled human serum albumin (HSA) to determine optimal PEGMA concentration...
January 11, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28077636/klebsiella-phage-%C3%AE-k64-1-encodes-multiple-depolymerases-for-multiple-host-capsular-types
#6
Yi-Jiun Pan, Tzu-Lung Lin, Ching-Ching Chen, Yun-Ting Tsai, Yi-Hsiang Cheng, Yi-Yin Chen, Pei-Fang Hsieh, Yi-Tsung Lin, Jin-Town Wang
: The genome of the multi-host bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5 was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional 8 putative capsule depolymerases (S2-4 against K1; S1-1 against K11; S1-3 against K21; S2-2 against K25; S2-6 against K30/K69; S2-3 against K35; S1-2 against KN4; S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins...
January 11, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28077445/human-antiviral-protein-ifix-suppresses-viral-gene-expression-during-hsv-1-infection-and-is-counteracted-by-virus-induced-proteasomal-degradation
#7
Marni S Crow, Ileana M Cristea
The interferon inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes antiviral cytokines expression. How IFIX exerts its host defense functions and whether it is inhibited by the virus remains unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection...
January 11, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28077248/alternative-splicing-at-exon-2-results-in-the-loss-of-the-catalytic-activity-of-mouse-dna-polymerase-iota-in-vitro
#8
Konstantin Y Kazachenko, Nataliya A Miropolskaya, Leonid V Gening, Vyacheslav Z Tarantul, Alena V Makarova
Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2...
January 4, 2017: DNA Repair
https://www.readbyqxmd.com/read/28072389/suppression-of-c9orf72-rna-repeat-induced-neurotoxicity-by-the-als-associated-rna-binding-protein-zfp106
#9
Barbara Celona, John von Dollen, Sarat C Vatsavayai, Risa Kashima, Jeffrey R Johnson, Amy A Tang, Akiko Hata, Bruce L Miller, Eric J Huang, Nevan J Krogan, William W Seeley, Brian L Black
Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS), but the mechanisms underlying repeat-induced disease remain incompletely resolved. One proposed gain-of-function mechanism is that repeat-containing RNA forms aggregates that sequester RNA binding proteins, leading to altered RNA metabolism in motor neurons. Here, we identify the zinc finger protein Zfp106 as a specific GGGGCC RNA repeat-binding protein, and using affinity purification-mass spectrometry, we show that Zfp106 interacts with multiple other RNA binding proteins, including the ALS-associated factors TDP-43 and FUS...
January 10, 2017: ELife
https://www.readbyqxmd.com/read/28070892/extraction-partial-purification-and-characterization-of-amylase-from-parthenocarpic-date-p-dactylifera-effect-on-the-cake-quality
#10
Hanen El Abed, Bassem Khemakhem, Imen Fendri, Mouna Chakroun, Mehdi Triki, Noureddine Drira, Hafedh Mejdoub
BACKGROUND: Phoenix dactylifera L. plays an important role in social, economic, and ecological Tunisian sectors. Some date palms produce parthenocarpic fruit named Sish. The objective of the present study is to extract biomolecules from parthenocarpic fruit by producing value-added products from the fruits. RESULTS: The optimization extraction of amylolytic activity from parthenocarpic fruit (AmyPF) was carried out using Box Behnken Design (BBD). A partial purification of about 250 folds with an activity yield of 47% was achieved...
January 10, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28070540/data-on-the-optimizations-of-expression-and-purification-of-human-bip-grp78-protein-in-escherichia-coli
#11
Jiao Yang, Lei Zhou, Qinglian Liu
Human BiP/GRP78 is involved in the folding and assembly of proteins in the endoplasmic reticulum. The proteins for crystallization in good amount and quality are prerequisites for obtaining ideal crystals. To meet these requirements, different BiP/GRP78 constructs, competent cells, vectors, and concentrations of inducer were tested in order to obtain soluble BiP/GRP78 protein with the highest amount and best purity. The BiP-T229A-L3,4'-Smt3 fusion protein was expressed in a soluble manner and finally purified with the highest purity using size exclusion chromatography, which was suitable for further protein crystallization...
February 2017: Data in Brief
https://www.readbyqxmd.com/read/28069171/steric-exclusion-chromatography-for-purification-of-cell-culture-derived-influenza-a-virus-using-regenerated-cellulose-membranes-and-polyethylene-glycol
#12
Pavel Marichal-Gallardo, Michael M Pieler, Michael W Wolff, Udo Reichl
Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration...
December 29, 2016: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28068333/bdf1-bromodomains-are-essential-for-meiosis-and-the-expression-of-meiotic-specific-genes
#13
Encar García-Oliver, Claire Ramus, Jonathan Perot, Marie Arlotto, Morgane Champleboux, Flore Mietton, Christophe Battail, Anne Boland, Jean-François Deleuze, Myriam Ferro, Yohann Couté, Jérôme Govin
Bromodomain and Extra-terminal motif (BET) proteins play a central role in transcription regulation and chromatin signalling pathways. They are present in unicellular eukaryotes and in this study, the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae. Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression, blocking cells at the exit from prophase, before the first meiotic division. This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes, including APC complex components...
January 9, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28067849/pnserpin-a-novel-serine-protease-inhibitor-from-extremophile-pyrobaculum-neutrophilum
#14
Huan Zhang, Rui Fei, Baigong Xue, Shanshan Yu, Zuoming Zhang, Sheng Zhong, Yuanqi Gao, Xiaoli Zhou
Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner...
January 7, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28067309/the-autophagy-elongation-complex-atg5-12-16l1-positively-regulates-hcv-replication-and-is-required-for-wild-type-membranous-web-formation
#15
Ahmed M Fahmy, Patrick Labonté
Hepatitis C virus (HCV) infection induces intracellular membrane rearrangements, thus forming a membranous web (MW) in which HCV replication and assembly occur. The HCV-induced MW is primarily composed of double membrane vesicles (DMVs) transfused by multi-membrane vesicles. The autophagy machinery has been proposed to participate in the formation of such vesicles. However, no clear evidence has been found linking autophagy to the formation of these DMVs. In this study, we evaluated the role of the autophagy elongation complex (ATG5-12/16L1) in HCV replication and MW formation...
January 9, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28065867/cloning-expression-purification-and-biophysical-analysis-of-two-putative-halogenases-from-the-glycopeptide-a47-934-gene-cluster-of-streptomyces-toyocaensis
#16
Tabata P Cardoso, Larissa A de Sá, Priscila Dos S Bury, Sair M C Pacheco, Marcio V B Dias
Glycopeptides are an important class of antibiotics used in the treatment of several infections, including those ones caused by methicillin resistant Staphylococcus aureus. Glycopeptides are biosynthesized by a Non Ribosomal Peptide Synthase (NRPS) and the resulting peptide precursors are decorated by several tailoring enzymes, such as halogenases and glycosyltransferases. These enzymes are important targets of protein engineering to produce new derivatives of known antibiotics. Herein, we show the production of two putative halogenases, denominated as StaI and StaK involved in the biosynthesis of the glycopeptide A47,934 in Streptomyces toyocaensis NRRL 15,009...
January 5, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28065317/preparation-of-centralspindlin-as-an-active-heterotetramer-of-kinesin-and-gtpase-activating-protein-subunits-for-in-vitro-structural-and-functional-assays
#17
M Mishima
Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065274/probing-the-activity-of-eukaryotic-rhomboid-proteases-in-vitro
#18
B Cordier, M K Lemberg
Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065273/screening-and-characterization-strategies-for-nanobodies-targeting-membrane-proteins
#19
S Veugelen, M Dewilde, B De Strooper, L Chávez-Gutiérrez
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065266/production-of-recombinant-rhomboid-proteases
#20
E Arutyunova, R Panigrahi, K Strisovsky, M J Lemieux
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA)...
2017: Methods in Enzymology
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