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Protein Expression and Purification

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
Ruchi Gupta, Tatiana P Soares da Costa, Pierre Faou, Con Dogovski, Matthew A Perugini
Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC, which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V...
January 11, 2018: Protein Expression and Purification
David Lopez-Tejedor, Jose M Palomo
A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 °C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity...
January 8, 2018: Protein Expression and Purification
Prachi Pathak, Ashu Kumar, Prabhu Prasad Sarangi, Sameer Bhagyawant, Duraipandian Thavaselvam
Brucellosis is a zoonotic disease caused by various species of the genus Brucella. The control of disease mainly depends on its accurate and early diagnosis. Culture methods employed for diagnosis are time consuming and require well equipped biosafety level 3 laboratories and hence serological tests are favored alternative for brucellosis diagnosis. At present serological diagnosis is based on LPS (lipopolysaccharide) which is less specific as the LPS antigen of Brucella species shows cross reactivity with other gram-negative bacteria...
January 3, 2018: Protein Expression and Purification
Meng-I Lin, Takashi Nagata, Masato Katahira
Oxidative enzymes of white-rot fungi play a key role in lignin biodegradation. Among those fungus, Ceriporiopsis subvermispora degrades lignin before cellulose in wood; C. subvermispora is the only fungus that secretes all known types of manganese peroxidases (CsMnPs). Utilization of lignin-degrading peroxidases has been limited so far due to the lack of efficient preparation methods and intensive characterization. In this study, we developed a highly efficient method to prepare active CsMnPs through soluble expression by E...
January 2, 2018: Protein Expression and Purification
Xiao-Dong Song, Chen-Jian Liu, Shi-Hao Huang, Xiao-Ran Li, En Yang, Yi-Yong Luo
Quorum sensing (QS) is a means of cell-to-cell communication that regulates, via small signalling molecules, expression of a series of genes and controls multicellular behaviour in many bacterial species. The enzyme S-ribosylhomocysteine lyase (LuxS) transforms S-ribosylhomocysteine (SRH) into 4, 5-dihydroxy-2, 3-pentanedione (DPD), the precursor of the interspecies QS signalling molecule autoinducer-2 (AI-2). In this study, two LuxS-coding genes, luxS1 and luxS2, with 70% sequence identity were isolated from Lactobacillus plantarum YM-4-3, and overexpressed in Escherichia coli BL21 (DE3), and the protein products were purified successfully...
January 2, 2018: Protein Expression and Purification
Roman S Esipov, Dmitry A Makarov, Vasily N Stepanenko, Maria A Kostromina, Tatyana I Muravyova, Yaroslav A Andreev, Igor A Dyachenko, Sergey A Kozlov, Evgeny V Grishin
APHC3 is an analgesic polypeptide that was found in the sea anemone (Heteractis crispa), and contains 56 amino acid residues. This polypeptide is of interest for the development of medications for diseases, associated with inflammatory or neuropathological processes, as well as its use as an analgesic. This work presents an innovative biotechnological method for APHC3 production. We have constructed a recombinant plasmid intended for biosynthesizing the fusion protein consisting of a chitin-binding domain, DnaB mini-intein from Synechocystis sp...
December 28, 2017: Protein Expression and Purification
Matsujiro Ishibashi, Ryo Kawanabe, Norie Amaba, Shigeki Arai, Fina Amreta Laksmi, Kenta Komori, Masao Tokunaga
Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [1] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability...
December 27, 2017: Protein Expression and Purification
Asmita D Pawar, Deepshikha Verma, Venu Sankeshi, Rajeev Raman, Yogendra Sharma
The reliability and accuracy of conformational or functional studies of any novel multidomain protein rely on the quality of protein. The bottleneck in structural studies with the complete Big_2 domain containing proteins like LigA, LigB or MpIBP is usually their large molecular size owing to their multidomain (>10-12 domains) architectures. Interestingly, a soil bacterium Paenarthrobacter aurescens TC1, harbours a gene that encodes a protein comprising of four predicted Big_2 domains. We report here the expression and purification of this novel, multiple Big_2 domains containing protein, Arig of P...
December 26, 2017: Protein Expression and Purification
Eriko Aoki, Kazuo Fujiwara, Akio Shimizu, Sayaka Takase-Yoden, Masamichi Ikeguchi
To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability...
December 25, 2017: Protein Expression and Purification
S Barathiraja, P A V Gangadhara, V Umapathi, H J Dechamma, G R Reddy
Interferon lambda-3 (IFNλ3) which is also known as IL28B is a member of type III Interferons which are structurally and genetically different from type I Interferons. These Interferons induce signal transduction pathways similar to type I Interferons which results in the activation of Interferon Stimulated Genes (ISGs). This group of Interferons are tissue specific and reported to have antiviral activity. In the present communication, we report the expression of bovine IFNλ3 gene (coding for the mature protein) in Pichia pastoris, purification of the expressed protein and evaluation of its biological activity...
December 23, 2017: Protein Expression and Purification
Spiro Pavlopoulos, Dimitrios N Pelekoudas, Othman Benchama, Catherine M Rawlins, Jeffrey N Agar, Jay M West, Michael Malamas, Nikolai Zvonok, Alexandros Makriyannis
N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) enzyme with a catalytic cysteine residue that has highest activity at acidic pH. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Inhibitors of NAAA have been shown to increase endogenous levels of PEA, and are of interest as potential treatments for inflammatory disorders and other maladies. Currently, there are no X-ray or NMR structures of NAAA available to inform medicinal chemistry...
December 15, 2017: Protein Expression and Purification
Mehdi Mostafaei, Seyed Nezamedin Hosseini, Maryam Khatami, Amin Javidanbardan, Abbas Akhavan Sepahy, Ebadullah Asadi
In the production process of recombinant Hepatitis B surface antigen (rHBsAg) various separation techniques are used to purify this virus-like particle (VLP). In this study, we developed antibody-conjugated super-paramagnetic Fe3O4/SiO2 core-shell nanoparticles as a highly selective method for isolation of expressed rHBsAg in yeast Pichia pastoris. For this purpose, first, iron oxide magnetic nanoparticles (MNPs) were prepared by co-precipitation method in alkali media and coated with silica. Then the surface was activated by amine groups and conjugated with oxidized antibodies...
December 12, 2017: Protein Expression and Purification
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
Ajay B Maghodia, Christoph Geisler, Donald L Jarvis
No abstract text is available yet for this article.
December 8, 2017: Protein Expression and Purification
Juliana Cotabarren, Mariana Edith Tellechea, Francesc Xavier Avilés, Julia Lorenzo, Walter David Obregón
The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var...
December 6, 2017: Protein Expression and Purification
Sergio Calderon-Salais, Prisiliana Velazquez-Bernardino, Victor E Balderas-Hernandez, Ana P Barba-de la Rosa, Antonio De Leon-Rodriguez
Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164aa) of human calreticulin (hCALR) and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a multi-copy strain was obtained using an in vitro multimerization approach...
December 5, 2017: Protein Expression and Purification
Aleksandra Woznicka-Misaila, Céline Juillan-Binard, Delphine Baud, Eva Pebay-Peyroula, Stéphanie Ravaud
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor...
December 4, 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
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
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