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https://www.readbyqxmd.com/read/29155755/in-vivo-detection-and-analysis-of-rb-protein-sumoylation-in-human-cells
#1
Fengxi Meng, Xiaofeng Li, Hui Ren, Jiang Qian
The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions...
November 2, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155263/identification-and-characterisation-of-the-epididymal-proteins-in-the-lizard-eutropis-carinata-reptilia-squamata-schneider-1801
#2
R Medini, M Bhagya, S Samson
Lizards are seasonal breeders. Cyclic reproductive nature makes lizard as a useful model for the study of the reproductively active protein secretions in the epididymis. During breeding season, the epididymides of the lizard secret proteins that mixes with the spermatozoa and create a favourable environment for sperm maturation. In this spectrum, the aim of this study is to identify and characterize proteins which are present in the lumen of the epididymis of the lizard, E. carinata during the active phase of reproduction...
November 16, 2017: General and Comparative Endocrinology
https://www.readbyqxmd.com/read/29155144/bay-60-2770-activates-two-isoforms-of-nitric-oxide-sensitive-guanylyl-cyclase-evidence-for-stable-insertion-of-activator-drugs
#3
Anne Sömmer, Peter Sandner, Sönke Behrends
Nitric oxide sensitive guanylyl cyclase (NOsGC), a hemoprotein and the major physiological receptor for nitric oxide (NO), is a heterodimer with the α1/β1 and α2/β1 isoforms known to be important for NO-signaling and conversion of GTP to cGMP in humans. Two innovative classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent sGC stimulators, that stimulate NOsGC directly and also increase the affinity towards NO, and the heme-independent sGC activators, that are thought to bind to oxidized and heme-free NOsGC in tissues exposed to oxidative stress...
November 15, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29154996/selective-binding-magnetic-separation-and-purification-of-histidine-tagged-protein-using-biopolymer-magnetic-core-shell-nanoparticles
#4
Yang Zhou, Dandan Yan, Shaofei Yuan, Yawen Chen, Emmanuella E Fletcher, Haifeng Shi, Bangxing Han
In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni(2+) nanoparticles. The Ni(2+) on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nanoparticles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified...
November 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/29154787/functionalization-of-paramagnetic-nanoparticles-for-protein-immobilization-and-purification
#5
Lara A B C Carneiro, Richard J Ward
A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe3O4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy...
November 14, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/29151220/high-efficiency-expression-of-yeast-derived-g-protein-coupled-receptors-and-19-f-labeling-for-dynamical-studies
#6
Libin Ye, Alexander P Orazietti, Aditya Pandey, R Scott Prosser
We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2-6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2-4, resulting in final functional production of 0.5-3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29151158/guidelines-to-reach-high-quality-purified-recombinant-proteins
#7
REVIEW
Carla Oliveira, Lucília Domingues
The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process...
November 18, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29150942/a-novel-epididymal-quiescence-factor-inhibits-sperm-motility-by-modulating-nos-activity-and-intracellular-no-cgmp-pathway
#8
Prasanta Ghosh, Sandipan Mukherjee, Arpita Bhoumik, Sandhya Rekha Dungdung
Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF was purified to 215 fold with 45% activity recovery...
November 18, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/29149997/surface-sieving-coordinated-imac-material-for-purification-of-his-tagged-proteins
#9
Senwu Li, Kaiguang Yang, Lukuan Liu, Baofeng Zhao, Yuanbo Chen, Xiao Li, Lihua Zhang, Yukui Zhang
Tailor-made materials for the purification of proteins with His-tag was designed through synergizing the selectivity of surface sieving and metal ion affinity. By excluding impurity proteins out of the surface polymer network, such materials could purify His-tagged proteins from the crude cell lysis with purity up to 90%, improved by 14% compared to that obtained by the commercial metal chelating affinity materials. This study might promote the His-tagged protein purification to a new level.
January 2, 2018: Analytica Chimica Acta
https://www.readbyqxmd.com/read/29149798/reconstructing-biosynthetic-pathway-of-the-plant-derived-cancer-chemopreventive-precursor-glucoraphanin-in-escherichia-coli
#10
Han Yang, Feixia Liu, Yin Li, Bo Yu
Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia coli via gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway...
November 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29147100/purification-and-identification-of-a-natural-antioxidant-protein-from-fertilized-eggs
#11
Shaohua Yang, Lulu Wang, Ying Wang, Xiaoqian Ou, Zhaoyuan Shi, Chongchong Lu, Wei Wang, Guoqing Liu
Fertilized hen eggs are rich in a variety of bioactive ingredients. In this study, we aimed to obtain an antioxidant protein from fertilized eggs and the radical scavenging abilities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), superoxide anion (O(2-)•) were used to evaluate the antioxidant activity of the purified protein. During 20 d of incubation, the radical scavenging ability of protein extracted from fertilized eggs exhibited significantly differences and the protein on day 16 showed higher antioxidant capacity...
2017: Korean Journal for Food Science of Animal Resources
https://www.readbyqxmd.com/read/29147098/purification-and-anti-pathogenic-properties-of-immunoglobulin-concentrates-from-porcine-blood
#12
Tae-Hwan Jung, Jae-Hwan Choi, Kyung-Chul Koh, Woo-Min Jeon, Kyoung-Sik Han
During slaughtering, animal blood is typically discarded, resulting in water pollution. However, this discarded blood has valuable components, such as immunoglobulin (Ig). Although several studies have been conducted to develop methods for effective recycling of slaughterhouse blood, they have not been commercially utilized in Korea. Here, we extracted an Ig-rich fraction from porcine blood that was then subjected to various in vitro tests, including pathogen growth inhibition, antigenic cross-reactivity, and anti-toxin activity...
2017: Korean Journal for Food Science of Animal Resources
https://www.readbyqxmd.com/read/29146152/improved-antifungal-activity-of-barley-derived-chitinase-i-gene-that-overexpress-a-32kda-recombinant-chitinase-in-escherichia-coli-host
#13
Nida Toufiq, Bushra Tabassum, Muhammad Umar Bhatti, Anwar Khan, Muhammad Tariq, Naila Shahid, Idrees Ahmad Nasir, Tayyab Husnain
Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene...
October 31, 2017: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
https://www.readbyqxmd.com/read/29143228/effect-of-exosome-biomarkers-for-diagnosis-and-prognosis-of-breast-cancer-patients
#14
REVIEW
M Wang, S Ji, G Shao, J Zhang, K Zhao, Z Wang, A Wu
PURPOSE: Exosomes are gradually detected as an indicator for diagnosis and prognosis of breast cancer in clinic and a systematic review was conducted. METHODS: A search for clinical studies published before July 1, 2017 was performed. Methods of exosome purification and identification from all studies were extracted. For diagnosis evaluation, the comparison of exosome biomarkers expression between breast cancer patients and healthy women was obtained; for prognosis prediction, the correlation between exosome biomarkers expression and chemotherapy resistance, overall survival (OS), disease-free survival (DFS), recurrence and metastasis of breast cancer was also extracted...
November 15, 2017: Clinical & Translational Oncology
https://www.readbyqxmd.com/read/29143175/vectors-for-expression-of-signal-peptide-dependent-proteins-in-baculovirus-insect-cell-systems-and-their-application-to-expression-and-purification-of-the-high-affinity-immunoglobulin-gamma-fc-receptor-i-in-complex-with-its-gamma-chain
#15
Le T M Le, Jens R Nyengaard, Monika M Golas, Bjoern Sander
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER)...
November 15, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29142209/fbxo4-mediated-degradation-of-fxr1-suppresses-tumorigenesis-in-head-and-neck-squamous-cell-carcinoma
#16
Shuo Qie, Mrinmoyee Majumder, Katarzyna Mackiewicz, Breege V Howley, Yuri K Peterson, Philip H Howe, Viswanathan Palanisamy, J Alan Diehl
The Fbxo4 tumour suppressor is a component of an Skp1-Cul1-F-box E3 ligase for which two substrates are known. Here we show purification of SCF(Fbxo4) complexes results in the identification of fragile X protein family (FMRP, Fxr1 and Fxr2) as binding partners. Biochemical and functional analyses reveal that Fxr1 is a direct substrate of SCF(Fbxo4). Consistent with a substrate relationship, Fxr1 is overexpressed in Fbxo4 knockout cells, tissues and in human cancer cells, harbouring inactivating Fbxo4 mutations...
November 16, 2017: Nature Communications
https://www.readbyqxmd.com/read/29141403/lipogels-for-encapsulation-of-hydrophilic-proteins-and-hydrophobic-small-molecules
#17
Celia C Homyak, Ann Fernandez, Mollie A Touve, Bo Zhao, Francesca Anson, Jeanne A Hardy, Richard W Vachet, Nathan C Gianneschi, Jennifer L Ross, S Thayumanavan
Lipid-polymer hybrid materials have the potential to exhibit enhanced stability and loading capabilities in comparison to parent liposome or polymer materials. However, complexities lie in formulating and characterizing such complex nanomaterials. Here we describe a lipid-coated polymer gel (lipogel) formulated using a one-pot methodology, where self-assembling liposomes template a UV-curable polymer gel core. Using fluorescently-labeled lipids, protein, and hydrophobic molecules, we have characterized their formation, purification, stability, and encapsulation efficiency via common instrumentation methods such as dynamic light scattering (DLS), matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), UV-Visible spectroscopy, fluorescence spectroscopy, and single particle total internal reflection fluorescence (TIRF) microscopy...
November 15, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/29139018/vaccination-against-hydatidosis-molecular-cloning-and-optimal-expression-of-the-eg95nc-recombinant-antigen-in-escherichia-coli
#18
M Jazouli, M Lightowlers, C G Gauci, K Tadlaoui, A Belmlih, M M Ennaji, M Elharrak
Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries...
November 14, 2017: Protein Journal
https://www.readbyqxmd.com/read/29138497/rapid-purification-of-human-bispecific-antibodies-via-selective-modulation-of-protein-a-binding
#19
Adam Zwolak, Catherine N Leettola, Susan H Tam, Dennis R Goulet, Mehabaw G Derebe, Jose R Pardinas, Songmao Zheng, Rose Decker, Eva Emmell, Mark L Chiu
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs...
November 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29138064/bicontinuous-microemulsions-as-a-biomembrane-mimetic-system-for-melittin
#20
Douglas G Hayes, Ran Ye, Rachel N Dunlap, Divina B Anunciado, Sai Venkatesh Pingali, Hugh M O'Neill, Volker S Urban
Antimicrobial peptides effectively kill antibiotic-resistant bacteria by forming pores in prokaryotes' biomembranes via penetration into the biomembranes' interior. Bicontinuous microemulsions, consisting of interdispersed oil and water nanodomains separated by flexible surfactant monolayers, are potentially valuable for hosting membrane-associated peptides and proteins due to their thermodynamic stability, optical transparency, low viscosity, and high interfacial area. Here, we show that bicontinuous microemulsions formed by negatively-charged surfactants are a robust biomembrane mimetic system for the antimicrobial peptide melittin...
November 11, 2017: Biochimica et Biophysica Acta
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