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Affinity chromatography

Spencer C Harris, Saravanan Devendran, João M P Alves, Sean M Mythen, Phillip B Hylemon, Jason M Ridlon
BACKGROUND: The multi-step bile acid 7α-dehydroxylating pathway by which a few species of Clostridium convert host primary bile acids to toxic secondary bile acids is of great importance to gut microbiome structure and host physiology and disease. While genes in the oxidative arm of the 7α-dehydroxylating pathway have been identified, genes in the reductive arm of the pathway are still obscure. METHODS: We identified a candidate flavoprotein-encoding gene predicted to metabolize steroids...
December 4, 2017: Biochimica et Biophysica Acta
Edita Voitechovič, Anton Korepanov, Dmitry Kirsanov, Andrey Legin
Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system...
December 4, 2017: Journal of Pharmaceutical and Biomedical Analysis
I Rodríguez-Ruiz, V Babenko, S Martínez-Rodríguez, J A Gavira
Lab-on-a-Chip (LoC), or micro-Total Analysis Systems (μTAS), is recognized as a powerful analytical technology with high capabilities, though end-user products for protein purification are still far from being available on the market. Remarkable progress has been achieved in the separation of nucleic acids and proteins using electrophoretic microfluidic devices, while pintsize devices have been developed for protein isolation according to miniaturized chromatography principles (size, charge, affinity, etc...
December 7, 2017: Analyst
Wu-Mei Yuan, Wan-Ju Zhang, Fen-Lian Ma, Jin-Song Li, Qian Zhang, Li-Shu Zheng
Background: Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor. Results: A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities...
2017: Cellular & Molecular Biology Letters
Hye Jin Kim, Kyou Hee Shim, Seung Ju Yeon, Hwa Sung Shin
Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator (t-PA) has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study was aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats...
December 8, 2017: Journal of Microbiology and Biotechnology
Marylane de Sousa, Ricardo M Manzo, José L García, Enrique J Mammarella, Luciana R B Gonçalves, Benevides C Pessela
l-Arabinose isomerase (EC (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics...
December 6, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Nataliya R Rovnyagina, Nikolai N Sluchanko, Tatiana N Tikhonova, Victor V Fadeev, Artur Yu Litskevich, Alexander A Maskevich, Evgeny A Shirshin
Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography...
December 2, 2017: International Journal of Biological Macromolecules
Yu-Jie Wang, Lin Li, Wei-Juan Song, Yan-Jun Zhou, Meng-Da Cao, Xiang-Rong Zuo, Ji-Fu Wei
Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test...
October 31, 2017: Oncotarget
Nives Ivic, Silvija Bilokapic, Mario Halic
H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1...
2017: PloS One
Yaofang Zhang, Li Du, Yanling Xu, Lin Tian, Peipei Tu, Minggang Li
BACKGROUND: Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) play a similar but complementary role in the regulation of glucose levels in islet β-cells. This study was aimed to develop a fusion peptide, which combines 4 tandem repeated GLP-1 and 4 tandem repeated GIP (4rolGG), and to investigate its therapeutic effect on type 2 diabetes using a diabetic mice model. METHOD: A 4rolGG expression plasmid was constructed and expressed in BL21 (DE3)...
December 3, 2017: Current Pharmaceutical Biotechnology
Qianni Liu, Meijuan Xu, Rongzhen Zhang, Meizhou Wang, Xian Zhang, Taowei Yang, Zhiming Rao
Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Huawei He, Yejing Wang, Kai Song, Jiao Wang, Shuguang Wei, Peng Zhao, Ping Zhao
Protein tyrosine phosphatase (PTP, EC specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Ayesha Siddiqui, Mustafa Kamal, Seyed Abdulmajid Ayatollahi, Mohsin Ali, Mansoor Ahmed
Bacillus licheniformis RM44 was isolated from hot spring near Karachi and screened for the production of extracellular amylase Amy RM44. Amy RM44 was purified to homogeneity on a single step by affinity chromatography using insoluble corn starch. The molecular weight of Amy RM44 was estimated to be 66 kDa by SDS-PAGE and zymographic analysis. Nine fold purification was achieved with the specific activity of 870 U/mg that provides the total yield of the enzyme up to 31%. Studies on purified AmyRM44 characterization revealed that the optimum temperature of enzyme was 100 ºC...
2017: Iranian Journal of Pharmaceutical Research: IJPR
Chenhua Zhang, Elliott Rodriguez, Cong Bi, Xiwei Zheng, Doddavenkatana Suresh, Kyungah Suh, Zhao Li, Fawzi Elsebaei, David S Hage
The last few decades have witnessed the development of many high-performance separation methods that use biologically related binding agents. The combination of HPLC with these binding agents results in a technique known as high performance affinity chromatography (HPAC). This review will discuss the general principles of HPAC and related techniques, with an emphasis on their use for the analysis of biological compounds and pharmaceutical agents. Various types of binding agents for these methods will be considered, including antibodies, immunoglobulin-binding proteins, aptamers, enzymes, lectins, transport proteins, lipids, and carbohydrates...
December 4, 2017: Analyst
Tifany Oulavallickal, Jodi L Brewster, James L O McKellar, Michael J Fairhurst, Nicholas A Tenci, Monica L Gerth
Nitrate- and nitrite-sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor-associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF-SD; residues 42-332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21-Gold(DE3) cells and purified via immobilized metal-affinity and size-exclusion chromatography...
December 1, 2017: Acta Crystallographica. Section F, Structural Biology Communications
Zhaoshou Yang, Wenjie Li, Zifan Yang, Aihua Pan, Wanqin Liao, Xingwang Zhou
Trichinellosis is a foodborne disease that remains a public health hazard and an economic problem in food safety. Vaccines against the parasite can be an effective way to control this disease; however, commercial vaccines against Trichinella infection are not yet available. Trichinella cathepsin B proteins appear to be promising targets for vaccine development. Here, we reported for the first time the characterization of a novel cDNA that encodes Trichinella spiralis (T. spiralis) cathepsin B-like protease 2 gene (TsCPB2)...
November 30, 2017: Vaccine
Adalgisa Wiecikowski, Katia Maria Dos Santos Cabral, Marcius da Silva Almeida, Renato Sampaio Carvalho
Galectin-3 (Gal3) is involved in many physiological processes related to tumor growth, such as promoting angiogenesis, cell migration/invasion, resistance to apoptosis and immune response modulation. Usually the overexpression of Gal3 is a poor prognostic marker for cancer patients. Recombinant Gal3 carbohydrate domain (Gal3C) has been proposed as a useful tool to inhibit angiogenesis. So far, all production protocols reported for Gal3C production have used proteolytic cleavage of full length Gal3 and/or affinity-based purification...
November 30, 2017: Protein Expression and Purification
Kate C Verbeeten, Alexandra H Ahmet
Cortisol is a hydrophobic molecule that is largely bound to corticosteroid-binding globulin (CBG) in the circulation. In the assessment of adrenal insufficiency, many clinicians measure a total serum cortisol level, which assumes that CBG is present in normal concentrations and with a normal binding affinity for cortisol. CBG concentration and affinity are affected by a number of common factors including oral contraceptive pills (OCPs), fever and infection, as well as rare mutations in the serine protease inhibitor A6 (SERPINA6) gene, and as such, total cortisol levels might not be the ideal way to assess adrenal function in all clinical circumstances...
December 1, 2017: Journal of Pediatric Endocrinology & Metabolism: JPEM
S Lalitha Gavya, Neha Arora, Siddhartha Sankar Ghosh
A paradigm shift towards fusion proteins to render multiple functionalities and applications on a single platform has been incurred in enzyme based diagnosis. Herein, we report development and systematic characterizations of glutathione-s-transferase (GST) and lactate dehydrogenase-A (hLDHA) in a fusion protein (GST-hLDHA) to achieve functional activities of GST and hLDHA simultaneously. The GST-pGEX-4 T -2 vector system was employed for cloning and purification of human lactate dehydrogenase A, utilizing the affinity based interaction between GST and GSH in column chromatography...
December 1, 2017: Preparative Biochemistry & Biotechnology
Alan Deng, Steven G Boxer
Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e. split GFP with a β-strand removed, that were found to behave differently in the presence of light...
December 1, 2017: Journal of the American Chemical Society
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