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Affinity purification recombinant protein

Hung Huy Nguyen, Alexander N Volkov, Guy Vandenbussche, Peter Tompa, Kris Pauwels
Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules...
January 12, 2018: Protein Expression and Purification
Chikako Ono, Junki Hirano, Toru Okamoto, Yoshiharu Matsuura
Insect expression systems based on baculovirus are widely used for the generation of recombinant proteins. Here, we evaluated the infectivity of baculoviruses under the physiological stresses of 'freeze- thaw' and sonication, and the baculoviral contamination of recombinant proteins after protein purification. Our findings suggest that Nonidet™ P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses...
January 11, 2018: Microbiology and Immunology
Honglian Wang, Xia Zhong, Jianchun Li, Menglian Zhu, Lu Wang, Xingli Ji, Junming Fan, Li Wang
IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H...
January 9, 2018: Molecular Biotechnology
Ana M Almeida, Joana Tomás, Patrícia Pereira, João A Queiroz, Fani Sousa, Ângela Sousa
DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology...
January 5, 2018: Biotechnology Progress
Stanislav S Beloborodov, Jiayin Bao, Svetlana M Krylova, Agnesa Shala-Lawrence, Philip E Johnson, Sergey N Krylov
DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state...
December 16, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Miguel A Lopez, Randi M Mackler, Matthew P Altman, Kristine E Yoder
The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer...
December 8, 2017: Journal of Visualized Experiments: JoVE
Sonu Bisht, Kumar Siddharth Singh, Ritu Choudhary, Sudarshan Kumar, Sunita Grover, Ashok Kumar Mohanty, Veena Pande, Jai Kumar Kaushik
The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form...
December 8, 2017: Protein Expression and Purification
Linpeng Wang, Jing Yan, Jingyun Yan, Huanhuan Xu, Dengyang Zhang, Xuanjun Wang, Jun Sheng
In the fields of drug discovery and protein science, small quantities of proteins are always needed to investigate or validate protein-protein (or protein-small molecule) interactions. Traditional transient or stable expression method to obtain recombinant proteins in eukaryotic systems can be laborious and time-consuming, especially when multiple protein variants are required. Here, we present a fast and convenient method for obtaining small quantities of recombinant human epidermal growth factor receptor (rhEGFR) ectodomain protein, which could be efficiently extended to the expression of other eukaryotic proteins...
December 4, 2017: Protein Expression and Purification
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
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
Liwei Yan, Wei Gong, Wenbing Zhu, Xuemei Zhang, Jingwen Xu, Zhongxiang Wu, Kongjie Lu, Ming Sun, Shaozhong Dong
We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells...
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
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
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
Carla Marusic, Claudio Pioli, Szymon Stelter, Flavia Novelli, Chiara Lonoce, Elena Morrocchi, Eugenio Benvenuto, Anna Maria Salzano, Andrea Scaloni, Marcello Donini
Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity...
November 27, 2017: Biotechnology and Bioengineering
Angela Huynh, Donald M Arnold, Jane C Moore, James W Smith, John G Kelton, Ishac Nazy
Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4...
November 27, 2017: Platelets
Martina Tuttolomondo, Pernille Lund Hansen, Jan Mollenhauer, Henrik J Ditzel
Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol...
November 20, 2017: Analytical Biochemistry
Brian S Hamilton, Joshua D Wilson, Marina A Shumakovich, Adam C Fisher, James C Brooks, Alyssa Pontes, Radnaa Naran, Christian Heiss, Chao Gao, Robert Kardish, Jamie Heimburg-Molinaro, Parastoo Azadi, Richard D Cummings, Judith H Merritt, Matthew P DeLisa
Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli...
November 21, 2017: Scientific Reports
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 18, 2017: Analytical Biochemistry
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]
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