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Affinity chromatography

Stefania Rosito, Grazia Paola Nicchia, Claudia Palazzo, Anna Lia, Cinzia Buccoliero, Francesco Pisani, Maria Svelto, Maria Trojano, Antonio Frigeri
Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO-IgG) against the water channel aquaporin-4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle...
October 20, 2017: Journal of Cellular and Molecular Medicine
Gérald Perret, Egisto Boschetti
The present review deals with the place of single chain oligonucleotide ligands (aptamers) in affinity chromatography applied to proteins. Aptamers are not the only affinity ligands available but they represent an emerging and highly promising route that advantageously competes with antibodies in immunopurification processes. A historical background of affinity chromatography from the beginning of the discipline to the most recent outcomes is first presented. Then the focus is centered on aptamers which represent the last step so far to the long quest for affinity ligands associating very high specificity, availability and strong stability against most harsh cleaning agents required in chromatography...
October 17, 2017: Biochimie
Zehui Zhang, Fan Wang, Chuandao Chen, Zhou Zheng, Jude Juventus Aweya, Yueling Zhang
Hemocyanin is an important multifunctional non-specific immune molecule. In this study, we purified lectin binding and non-lectin binding hemocyanin from Litopenaeus vannamei using Concanavalin A (ConA) lectin affinity chromatography (designated HMC-C and HMC-NC, respectively). Analysis of the carbohydrate content showed that HMC-C had about 20 times as much carbohydrate as HMC-NC. 54 and 42 peaks were observed in HMC-C and HMC-NC by HPLC, which reduced to 49 and 6 peaks, respectively, when digested with trypsin and repurified with ConA lectin column...
October 16, 2017: Immunology Letters
Weiwei Zheng, Liu Yang, Chenggu Cai, Jinfeng Ni, Bo Liu
The sweet protein monellin has high sweet potency with limited stability. In this study, 3 double-sites mutants (E2N/E23A, E2N/Y65R and E23A/Y65R) of the single-chain monellin (MNEI) were constructed. The proteins were expressed in E. coli BL21 and purified to homogeneity by nickel affinity chromatography with yields above 10 mg/L cell culture. Introduction of a sweeter mutant E2N into E23A or Y65R (E2N/E23A and E2N/Y65R) led to about 3-fold increase of sweetness, while addition of a more stable mutant E23A into E2N or Y65R (E2N/E23A and E23A/Y65R) resulted in improved thermal stability (about 10 °C)...
October 16, 2017: Protein Expression and Purification
Xudong Sun, Lingyi Zhang, Weibing Zhang
Because of the low abundance of glycoprotein and glycopeptide in complex biological samples, it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research. Herein, a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO2). The developed hydrophilic silica was packed into a solid phase extraction (SPE) column, and applied to the highly selective enrichment and identification of N-linked glycopeptides...
July 8, 2017: Se Pu, Chinese Journal of Chromatography
Zhijun Li, Kim A Munro, Mina Narouz, Bin Hao, Cathleen M Crudden, J Hugh Horton, Hongxia Hao
Surface chemistry is a key enabler for various biosensing applications. Surface plasmon resonance (SPR)-based biosensors routinely employ thiol-based chemistry for the linker layer between gold-coated support surfaces and functional biosensor surfaces. However, there is a growing awareness that such sensor surfaces are prone to oxidation/degradation problems in the presence of oxygen, and previous efforts to improve the stability have shown limited advancements. As an alternative, recent studies employing N-heterocyclic carbene (NHC) self assembled monolayers (SAMs) deposited on gold have shown significant promise in this area...
October 19, 2017: ACS Applied Materials & Interfaces
Yi Wu, Min Yu, Zujun Sun, Weihua Hou, Yuxiong Wang, Qingyun Yuan, Wei Mo
Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. The goal of this study was to create a novel bispecific antibody targeting both cellular-mesenchymal to epithelial transition factor(c-MET) and programmed death-1(PD-1) as an anti-cancer therapeutic candidate. Upon binding to the c-MET antigen on a tumor cell and the PD-1 antigen on a T cell, a BsAb can redirect T cells for tumor killing. Based on the original sequences of PD-1 and c-MET mAbs, a BsAb gene was designed, and cloned into the pCEP4 vector...
October 17, 2017: Protein and Peptide Letters
Yuxiao Yuan
The objective of this paper is to study the global DNA Methylation in monozygotic (MZ) twins with cerebral palsy. Two pairs of twins (a cerebral palsy children, a normal child) admitted to the First Affiliated Hospital of Zhengzhou University were selected as subjects. The phenol-chloroform method was used to extract DNA from venous blood and micro satellite DNA genotyping technique was used to identify the eggs of the twins. DNA methylation fragments were enriched by MBD affinity column chromatography, followed by Solexa sequencing and bioinformatics analysis...
July 2017: Pakistan Journal of Pharmaceutical Sciences
Nelson C N Chan, King-Ho Chow, Rico F Y Leung, Sequence S H Tang, Michelle F W Chiu, Rosalind Lie, Kelvin C K Cheng, Kin-Mang Lau, Natalie P H Chan, Margaret H L Ng
We report a novel HBB: c.114G>C mutation in a Chinese family. This mutation resulted in a β37(C3)Trp→Cys amino acid substitution and was synonymous with Hb Kent, a hemoglobin (Hb) variant that was reported exclusively in patients of European descent. Though Hb Kent has a normal oxygen affinity and molecular stability, it has a characteristic dual variant appearance on cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC) caused by the posttranslational modification of cysteine...
October 18, 2017: Hemoglobin
Min Jiang, Lujia Zhang, Fengqing Wang, Jie Zhang, Guosong Liu, Bei Gao, Dongzhi Wei
Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the magnetic protein was firstly used in biotechnology application, and a novel convenient one-step purification and immobilization method was established. A universal vector and three linker patterns were developed for fusion expression of magnetic protein and target protein. The magnetic protein was absorbed by iron beads, followed by target protein aggregation, purification, and immobilization...
October 17, 2017: Scientific Reports
Patil Armenian, Kathy T Vo, Jill Barr-Walker, Kara L Lynch
Deaths from opioid use are increasing in the US, with a growing proportion due to synthetic opioids. Until 2013, sporadic outbreaks of fentanyl and fentanyl analogs contaminating the heroin supply caused some deaths in heroin users. Since then, fentanyl has caused deaths in every state and fentanyl and its analogs have completely infiltrated the North American heroin supply. In 2014, the first illicit pills containing fentanyl, fentanyl analogs, and other novel synthetic opioids such as U-47700 were detected...
October 14, 2017: Neuropharmacology
Bi Xu, Kuixiao Li, Juan Qiao, Zhiqi Liungai, Chao Chen, Yun Lu
As one of the most oestrogenic synthetic compounds in water environment, diethylstilbestrol (DES) has been studied for decades. Some studies showed that DES can be removed by ultraviolet (UV) irradiation. However, no one has paid attention to the formation of oestrogenic disinfection by-products (DBPs) and the persistence of DES in surface water remains a mystery. In this study, UV was found to be very effective in removing oestrogenic activity regardless of water quality. Three oestrogenic DBPs were specifically isolated by oestrogen receptor-based affinity chromatography and identified as 9,10-diethylphenanthrene-3,6-diol, cis-DES and Z,Z-dienestrol...
October 3, 2017: Water Research
Chen Cui, Ligang Huang, Jing Li, Xingqi Zou, Yuanyuan Zhu, Lei Xie, Qizu Zhao, Limin Yang, Wenjun Liu
Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively...
November 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Andrew J Borst, Zachary M James, William N Zagotta, Mark Ginsberg, Felix A Rey, Frank DiMaio, Marija Backovic, David Veesler
The LM609 antibody specifically recognizes αVβ3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVβ3-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αVβ3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVβ3...
October 7, 2017: Structure
Bich Hang Do, Minh Tan Nguyen, Jung-A Song, Sangsu Park, Jiwon Yoo, Jaepyeong Jang, Sunju Lee, Seoungjun So, Yejin Yoon, Inki Kim, Kyungjin Lee, Yeon Jin Jang, Han Choe
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an anti-tumor agent due to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield due to its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase (PDI) at the N-terminus of TRAIL...
October 14, 2017: Journal of Microbiology and Biotechnology
Yanjun Ding, Xingmei Li, Yadong Guo, Jie Yan, Jiang Ling, Weichen Li, Lingmei Lan, Yunfeng Chang, Jifeng Cai, Lagabaiyla Zha
In recent years, drug abuse has been considered as a most challenging social problem that aroused public attention. Ketamine has increased in unregulated use as a 'recreational drug' in teenagers. However, there is no suitable and maneuverable detection method for ketamine in situ at the moment. Fluorescence sensor technique, with predominant recognition and simple operation, is a good potential application in drug detection. Here, we first reported a highly sensitive and selective fluorescence genosensor for rapid detection of ketamine based on DNA-templated silver nanoclusters (DNA-AgNCs) probes, in which the DNA sequence could specially recognize ketamine with high affinity...
October 14, 2017: Analytical and Bioanalytical Chemistry
R Vijayakumar, Timir Tripathi
We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction...
October 12, 2017: Protein Expression and Purification
Mingyu Zhang, Gong Li, Yi Zhang, Jingwu Kang
The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs...
October 6, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Gabriel Ramos Ferreira Gonçalves, Olga Reinert Ramos Gandolfi, Leandro Soares Santos, Renata Cristina Ferreira Bonomo, Cristiane Martins Veloso, Lizzy Ayra Alcântara Veríssimo, Rafael da Costa Ilhéu Fontan
Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance...
October 9, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Harkiranpreet Kaur Dhaliwal, Nagarajan Thiruvanakarasu, Raj Kumar, Kruti Patel, Ghuncha Ambrin, Shouwei Cai, Bal Ram Singh
Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively...
October 13, 2017: Protein Journal
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