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Affinity chromatography

Chunyan Liu, Gen Zou, Xing Yan, Xuguo Zhou
Termite gut microbiome is a rich reservoir for glycoside hydrolases, a suite of enzymes critical for the degradation of lignocellulosic biomass. To search for hemicellulases, we screened 12,000 clones from a fosmid gut library of a higher termite, Globitermes brachycerastes. As a common Southeastern Asian genus, Globitermes distributes predominantly in tropical rain forests and relies on the lignocellulases from themselves and bacterial symbionts to digest wood. In total, 22 positive clones with β-xylosidase activity were isolated, in which 11 representing different restriction fragment length polymorphism (RFLP) patterns were pooled and subjected to 454 pyrosequencing...
2018: International Journal of Biological Sciences
Justin M Choi, Valerie Bourassa, Kevin Hong, Michael Shoga, Elizabeth Lim, Andrew Park, Kazim Apaydin, Andrew K Udit
The potential applications for nanomaterials continue to grow as new materials are developed and environmental and safety concerns are more adequately addressed. Virus-like particles (VLPs) in particular have myriad applications in medicine and biology, exploiting both the reliable, symmetric self-assembly mechanism and the ability to take advantage of surface functionalities that may be appropriately modified through mutation or bioconjugation. Herein we describe the design and application of hybrid VLPs for use as potent heparin antagonists, providing an alternative to the toxic heparin antidote protamine...
June 14, 2018: Molecular Pharmaceutics
Syed Azmal Ali, Dhruba Malakar, Jai Kumar Kaushik, Ashok Kumar Mohanty, Sudarshan Kumar
Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects in vivo and in vitro, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS)...
2018: PloS One
Eliza Blicharska, Małgorzata Tatarczak-Michalewska, Anita Plazińska, Wojciech Plaziński, Anna Kowalska, Anna Madejska, Monika Szymańska-Chargot, Anna Sroka-Bartnicka, Jolanta Flieger
Spinacia oleracea L. extract was immobilized on an octadecyl-bonded silica surface to produce a new sorbent for the solid-phase extraction of trace amounts of metal ions from aqueous neutral samples. A measurement of the metal content has been performed by using graphite furnace atomic absorption spectroscopy. The affinity of the investigated bivalent metal cations for the modified sorbent are in the order: Pb(II) > Cu(II) > Ni(II) > Zn(II) ≈ Cd(II) ≈ Co(II). The quantum-chemically calculated chlorophyll-a-metal ion binding energies were consistent with the measured affinities of the corresponding metal ions to the investigated sorbent...
June 13, 2018: Journal of Separation Science
Xiao-Lin Ao, Xi Yu, Ding-Tao Wu, Chao Li, Tong Zhang, Shu-Liang Liu, Shu-Juan Chen, Li He, Kang Zhou, Li-Kou Zou
The strain Y1, with a notably high production of neutral protease, was isolated from naturally fermented broad beans and subsequently identified as Aspergillus oryzae, through the analysis of its morphology characteristics and 18S rDNA sequence. Naturally fermented broad beans are the main raw material in Sichuan broad-bean sauce. The neutral protease from Aspergillus oryzae Y1 was purified using ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography, which resulted in a 10.0-fold increase in the specific activity (2264...
June 12, 2018: AMB Express
B B Gengenbach, C R Müschen, J F Buyel
Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones...
June 9, 2018: Protein Expression and Purification
Zhan He, Hang Wang, Xiao Han, Tiantian Ma, Yi Hang, Huifei Yu, Fangfang Wei, Jing Sun, Yongchun Yang, Changyong Cheng, Houhui Song
We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711...
May 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Haibin Tong, Siya Wu, Kangxing Song, Jian Liu, Xindan Song, Xu Zhang, Luqi Huang, Mingjiang Wu
P-selectin is a promising therapeutic target for acute inflammation-related diseases, and interest has been growing in the search for high-affinity glycoconjugate ligands that can target the initial P-selectin-mediated recruitment of neutrophils to the site of inflammation. In our previous study, we isolated a water-soluble polysaccharide (BCPS) from Bupleurum chinense and showed that it exhibits anti-inflammatory effect by antagonizing P-selectin-mediated adhesion of HL-60 cells to CHO-P cells. In this study, we prepared a P-selectin-based affinity chromatography medium and used it to purify the P-selectin-binding moiety of BCPS...
September 15, 2018: Carbohydrate Polymers
Abass Tanhaiean, Marjan Azghandi, Jamshid Razmyar, Elyass Mohammadi, Mohammad Hadi Sekhavati
Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro...
June 8, 2018: Microbial Pathogenesis
Mark B Zimering
Aims: To test whether circulating neurotoxic autoantibodies increase in adult type 2 diabetes mellitus with Parkinson's disease (PD) or dementia. To identify the G-protein coupled receptor on neuroblastoma cells mediating neural inhibitory effects in diabetic Parkinson's disease plasma autoantibodies. To determine the mechanism of accelerated neuroblastoma cell death and acute neurite retraction induced by diabetic Parkinson's disease and dementia autoantibodies. Methods: Protein-A eluates from plasma of twelve older adult male diabetic patients having Parkinson's disease (n=10) or dementia (n=2), and eight age-matched control diabetic patients were tested for ability to cause accelerated N2A neuroblastoma cell death and acute neurite retraction...
2018: Journal of Endocrinology and Diabetes
F Verachi, Z Percario, P Di Bonito, E Affabris, C Amici, L Accardi
In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models...
2018: BioMed Research International
Dominik Buschmann, Benedikt Kirchner, Stefanie Hermann, Melanie Märte, Christine Wurmser, Florian Brandes, Stefan Kotschote, Michael Bonin, Ortrud K Steinlein, Michael W Pfaffl, Gustav Schelling, Marlene Reithmair
Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications...
2018: Journal of Extracellular Vesicles
Elentina K Argyrousi, Agnieszka Staniszewski, Russell E Nicholls, Ottavio Arancio
Oligomerization of soluble tau protein is attracting the attention of an increasingly larger number of scientists involved in research on Alzheimer's disease and other tauopathies. A variety of methods have been developed for the purification of proteins from biological tissues and bacterial cells. Various types of high performance liquid chromatography (HPLC) and affinity tags represent the most common techniques for isolating proteins. Here, we describe a procedure for extracting recombinant tau protein from bacterial cells, utilizing a 6×His affinity tag, or endogenous tau from brain cortices using acid extraction followed by fast protein liquid chromatography (FPLC)...
2018: Methods in Molecular Biology
Linfang Chen, Liujun Xue, Jinlong Zheng, Xiangyang Tian, Yingdong Zhang, Qiang Tong
Recent studies have indicated that peroxisome proliferator-activated receptor β/δ (PPARß/δ) agonists exert neuroprotective effects in the model of Parkinson's disease (PD). Furthermore, PPARß/δ agonists have been shown to have potential anti-inflammatory activity, but the underlying mechanisms remain obscure. Emerging evidence indicates that the nucleotide-binding domain and leucine-rich-repeat-protein 3 (NLRP3) inflammasome-mediated neuroinflammation plays a crucial role in the pathogenesis of PD. In the present study we investigate whether PPARß/δ agonists alleviate NLRP3-mediated neuroinflammation in the 1- methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model of PD...
June 7, 2018: Behavioural Brain Research
Jongho Jeon, Jiwon Yang, Jong-Moon Park, Na-Young Han, Yeong-Bae Lee, Hookeun Lee
Advanced glycation end products (AGEs) are known to play a leading part in the pathogenesis of human diseases, such as diabetes, Alzheimer's disease, lateral sclerosis, and atherosclerosis. It is for this reason that research on AGEs is crucial and large-scale studies are needed for the treatment of diseases. The aim of this study was to develop a reproducible method to analyze Amadori compounds using an automated enrichment protocol for high-throughput analysis of clinical samples. The developed method enabled the enrichment of Amadori compounds simultaneously from 96 samples, and it was applied to the discovery of biomarkers in AGEs related diseases...
May 25, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Xue-Jun Zhang, Hai-Ling Li, Da-Yi Deng, Chong Ji, Xiao-Dong Yao, Jia-Xin Liu
Tetanus is still a major cause of human deaths in several developing countries. In particular, the neonatal form remains a significant public health problem. According to the World Health Organization, administration of tetanus toxoid is recommended for neonatal tetanus patients. Furthermore, tetanus antitoxin or anti-tetanus immunoglobulin (Ig) are used for mild case or intensive care. This paper discusses a novel purification technique for improving equine anti-tetanus Ig production. First, equine plasma dealt with two steps salting out with ammonium sulfate; second, ultrafiltration concentration liquid purified by one successive protein G based affinity chromatography steps; finally, the purified F(ab')2 fragments was characterized using biochemical and proteomic methods and shown to be pure and homogeneous...
May 29, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Kou Motani, Hidetaka Kosako
Protein phosphorylation is one of the most common post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by more than 500 genes in higher eukaryotes and play central roles in various cellular signaling pathways. Consequently, genetic abnormalities of protein kinases have been implicated in many diseases. To fully understand the complex phosphorylation-mediated signaling networks, it is important to globally identify and functionally characterize in vivo substrates of individual protein kinases...
June 5, 2018: Biochimica et Biophysica Acta
Qian Li, Xiaohui Ning, Yuxin An, Brett J Stanley, Yuan Liang, Jing Wang, Kaizhu Zeng, Fuhuan Fei, Ting Liu, Huanmei Sun, Jiajun Liu, Xinfeng Zhao, Xiaohui Zheng
Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta2-adrenoceptor (β2-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations; we achieved the best adsorption models for the binding of salbutamol, terbutaline and pseudoephedrine to immobilized β2-AR...
June 8, 2018: Analytical Chemistry
Shridhar Bale, Alexandra Martiné, Richard Wilson, Anna-Janina Behrens, Valérie Le Fourn, Natalia de Val, Shailendra K Sharma, Karen Tran, Jonathan L Torres, Pierre-Alain Girod, Andrew B Ward, Max Crispin, Richard T Wyatt
Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1...
2018: Frontiers in Immunology
Qing Zhao, Tian Zhu Jia, Qi Chen Cao, Fang Tian, Wan Tao Ying
The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N -glycosylation of key proteins. To examine the alterations of N -glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific N -glycosylation analysis of left ventricular cardiomyocyte proteins...
June 7, 2018: International Journal of Molecular Sciences
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