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Affinity chromatography

Diego A Díaz-Dinamarca, José I Jerias, Daniel A Soto, Jorge A Soto, Natalia V Díaz, Yessica Y Leyton, Rodrigo A Villegas, Alexis M Kalergis, Abel E Vásquez
Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity...
February 13, 2018: Molecular Biotechnology
Alessandra Altamirano, Andreas Naschberger, Barbara G Furnrohr, Radka Saldova, Weston B Struwe, Patrick M Jennings, Silvia Millán Martín, Suzana Malic, Immanuel Plangger, Stefan Lechner, Reina Pisano, Nicole Peretti, Bernd Linke, Mario M Aguiar, Friedrich Fresser, Andreas Ritsch, Tihana Lenac Rovis, Christina Goode, Pauline M Rudd, Klaus Scheffzek, Bernhard Rupp, Hans Dieplinger
Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypo-glycosylated forms were expressed in CHO, CHO-Lec1 and HEK293T cells. Fully non-glycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin...
February 14, 2018: Journal of Proteome Research
Javier E Ortiz, Adriana Garro, Natalia B Pigni, María Belén Agüero, German Roitman, Alberto Slanis, Ricardo D Enriz, Gabriela E Feresin, Jaume Bastida, Alejandro Tapia
BACKGROUND: In Argentina, the Amaryllidaceae family (59 species) comprises a wide variety of genera, only a few species have been investigated as a potential source of cholinesterases inhibitors to treat Alzheimer disease (AD). PURPOSE: To study the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of the basic dichloromethane extracts (E) from Hieronymiella aurea, H. caletensis, H. clidanthoides, H. marginata, and H. speciosa species, as well as the isolated compounds from these plant extracts...
January 15, 2018: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
Junjie Ao, Jiaping Gu, Tao Yuan, Dan Li, Yuning Ma, Zhemin Shen
A highly selective sample cleanup method using molecularly imprinted polymers (MIP) was developed for the enrichment of domoic acid (DA, an amnesic shellfish toxin) from both seawater and shellfish samples. Molecular modelling was firstly applied to screening a suitable functional monomer and optimize the polymer preparation. Theoretical results were in a good agreement with those of the experimental studies. MIP was prepared by precipitation polymerization using 1, 3, 5-pentanetricarboxylic acid and 2-(Trifluoromethyl)acrylic acid as the template molecule and functional monomer, respectively...
February 3, 2018: Chemosphere
Noraziah Nordin, Nazia Abdul Majid, Rozana Othman, Fatima Abdelmutaal Ahmed Omer, Muhammad Nazil Afiq Nasharuddin, Najihah Mohd Hashim
Plagioneurin B belongs to acetogenin group has well-established class of compounds. Acetogenin group has attracted worldwide attention in the past few years due their biological abilities as inhibitors for several types of tumour cells. Plagioneurin B was isolated via conventional chromatography and tested for thorough mechanistic apoptosis activity on human ovarian cancer cells (CAOV-3). Its structure was also docked at several possible targets using Autodock tools software. Our findings showed that plagioneurin B successfully inhibits the growth of CAOV-3 cells at IC50 of 0...
February 12, 2018: Apoptosis: An International Journal on Programmed Cell Death
Vishakha A Pawar, Anil S Prajapati, Rekha C Akhani, Darshan H Patel, R B Subramanian
A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni-NTA His affinity chromatography achieved a yield of 38...
February 2018: 3 Biotech
Yiqin Xie, Qianjing Liu, Yan Li, Chunhui Deng
Boronic acid affinity chromatography (BAAC) is one of the most significant methods in glycoproteomics research due to its low bias towards glycopeptides and easy enrichment process. In this work, core-shell structured magnetic metal-organic framework (MOF) composites with abundant boronic acid groups were designed and synthesized for selective glycopeptide enrichment based on BAAC. The as-prepared core-shell structured magnetic MOF composites (denoted as Fe3O4@PVP/PEI@MOF (B)) inherited strong magnetic responsiveness from the Fe3O4 core as well as ultrahigh surface area and abundant boronic acid sites from the MOF shell...
February 6, 2018: Journal of Chromatography. A
Andrea Speltini, Francesca Merlo, Federica Maraschi, Michela Sturini, Matteo Contini, Nicola Calisi, Antonella Profumo
Pristine humic acids (HAs) were thermally condensed onto silica microparticles by a one-pot, inexpensive and green preparation route obtaining a mixed-mode sorbent (HA-C@silica) with good sorption affinity for glucocorticoids (GCs). The carbon-based material, characterized by various techniques, was indeed applied as the sorbent for fixed-bed solid-phase extraction of eight GCs from river water and wastewater treatment plant effluent, spiked at different concentration levels in the range 1-400 ng L-1. After sample extraction, the target analytes were simultaneously and quantitatively eluted in a single fraction of methanol, achieving enrichment factor 4000 and 1000 in river water and wastewater effluent, respectively...
February 5, 2018: Journal of Chromatography. A
Yeong-Jun Park, Byung Kwon Jung, Sung-Jun Hong, Gun-Seok Park, Jerald Conrad Ibal, HuyQuang Pham, Jae-Ho Shin
The enzyme xylose isomerase (EC, XI) is responsible for theconversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studiedthe characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, a gene coding for XI (xylA) was insertedinto the pET-21a (+) expression vector and the construct was transformed into an Escherichia coli competent cell BL21 (DE3)...
February 13, 2018: Journal of Microbiology and Biotechnology
Elena P Kiseleva, Konstantin I Mikhailopulo, Evelina L Zdorovenko, Yuriy A Knirel, Galina I Novik
Role of microorganisms in induction of/protection from autoimmune diseases is proven though molecular mechanisms and bacterial/viral/yeast biopolymers responsible for these effects are in the research stage. Autoantobodies (AAbs) to thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg) as well as AAbs to transglutaminase 2 (anti-TG2) and antibodies to gliadins (anti-gliadins) are serological markers of autoimmune thyroid disease and celiac disease, respectively, and players in pathogenesis of these autoimmune diseases...
December 20, 2017: Carbohydrate Research
Zahra Bahadori, Hamid Reza Kalhor, Seyed Javad Mowla
Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P...
April 2018: Enzyme and Microbial Technology
Koki Sakagami, Toshihiro Masuda, Kenichi Kawano, Shiroh Futaki
All-hydrocarbon stapled peptides are a promising class of protein-protein interaction regulators; their potential therapeutic benefit arises because they have high binding affinity and specificity to intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides...
February 8, 2018: Molecular Pharmaceutics
Andrés Felipe Carrillo Rincón, Vasilka Magdevska, Luka Kranjc, Štefan Fujs, Rolf Müller, Hrvoje Petković
Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds 'Generally Recognised As Safe' status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium...
February 7, 2018: Applied Microbiology and Biotechnology
Wenjie Di, Wu Qu, Runying Zeng
AgaM1, a β-agarase belonging to glycoside hydrolases family 16 (GH16), was cloned from the environmental DNA of mangrove sediments. The gene agaM1 is 2136 bp in length and encodes a protein of 712 amino acids. The properties of recombinant AgaM1 (rAgaM1) were studied using prokaryotic expression. The optimum temperature and pH were 50 °C and 7.0, respectively, and rAgaM1 exhibited a high adaptability to wide ranges of temperature and pH. A relatively high activity was retained at from 30 to 60 °C and from pH 6...
February 7, 2018: Journal of Basic Microbiology
Jasmine M Corbin, Muchena J Kailemia, C Linn Cadieux, Salem Alkanaimsh, Kalimuthu Karuppanan, Raymond L Rodriguez, Carlito B Lebrilla, Douglas M Cerasoli, Karen A McDonald, Somen Nandi
Recombinant butyrylcholinesterase produced in a metabolically-regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing-friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE)...
February 7, 2018: Biotechnology and Bioengineering
Timothy S Collier, Zhicheng Jin, Celalettin Topbas, Cory Bystrom
Isolation of High Density Lipoproteins (HDL) for structural and functional studies typically relies on ultracentrifugation techniques which are time consuming and difficult to scale. With emerging interest in the clinical relevance of HDL structure and function to cardiovascular disease, a significant gap exists between current and desirable sample preparation throughput. To enable proteomic studies of HDL with large clinical cohorts, we have developed an affinity enrichment approach which relies on the association of histidine-tagged, lipid free ApoA-I with HDL followed by standard metal chelate chromatography...
February 7, 2018: Journal of Proteome Research
Aiganym T Zhumabek, Laura S Abeuova, Nurzhan S Mukhametzhanov, Herman B Scholthof, Yerlan M Ramankulov, Shuga A Manabayeva
Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle...
February 1, 2018: Journal of Virological Methods
Mark T Agasid, Xuemin Wang, Yiding Huang, Colleen M Janczak, Robert Branström, S Scott Saavedra, Craig A Aspinwall
The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6...
January 30, 2018: Protein Expression and Purification
Pingyang Tao, Saumen Poddar, Zuchen Sun, David S Hage, Jianzhong Chen
Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions...
January 31, 2018: Methods: a Companion to Methods in Enzymology
Dalgys Martínez, Jose Fernando Cantillo, Helber Herazo, Judith Wortmann, Walter Keller, Luis Caraballo, Leonardo Puerta L
BACKGROUND: Sensitization to allergens of the house dust mites Dermatophagoides pteronyssinnus and Blomia tropicalis is an important risk factor for asthma and allergic diseases. Allergen-specific immunotherapy is currently based on natural allergen extracts, however, in the last years recombinant allergens with different modifications have shown promising immunological properties that may be advantageously applied for developing novel allergy vaccines. METHODS: A hybrid molecule (MAVAC-BD-2) containing epitopes of B...
February 3, 2018: Immunology Letters
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