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Affinity purification recombinant protein

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 14, 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]
M Jazouli, M Lightowlers, C G Gauci, K Tadlaoui, A Belmlih, M M Ennaji, M Elharrak
Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries...
November 14, 2017: Protein Journal
Natalia I Faccinetti, Luciano L Guerra, Adriana V Sabljic, Silvina S Bombicino, Bruno D Rovitto, Ruben F Iacono, Edgardo Poskus, Aldana Trabucchi, Silvina N Valdez
BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography...
November 13, 2017: Microbial Cell Factories
Lise Schoonen, Kayleigh S van Esterik, Chunqiu Zhang, Rein V Ulijn, Roeland J M Nolte, Jan C M van Hest
Hexahistidines are very common tags used in the affinity chromatography purification of recombinant proteins. Although these tags are solely applied for their metal-binding properties, we found that they are also able to perform ester hydrolysis when attached to a protein. For instance, green fluorescent protein (GFP) and the cowpea chlorotic mottle virus (CCMV) are able to perform catalysis after introduction of the His-tag. By attaching a His-tag to an enzyme, a dual-functional catalyst was created, that can perform a two-step cascade reaction...
November 7, 2017: Scientific Reports
Julia Hennicke, Anna Maria Lastin, David Reinhart, Clemens Grünwald-Gruber, Friedrich Altmann, Renate Kunert
Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity...
November 4, 2017: Analytical Biochemistry
Guo-Li Gong, Yuan Wei, Zhong-Zhong Wang
In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified and characterized. The peptide MLH was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37 and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3)...
November 3, 2017: Preparative Biochemistry & Biotechnology
Srinivas Jayanthi, Ravi Kumar Gundampati, Thallapuranam Krishnaswamy Suresh Kumar
Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest...
November 1, 2017: Current Protocols in Protein Science
Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy
At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein...
October 2017: PLoS Pathogens
Andrea Colarusso, Marco Caterino, Alessia Fabbri, Carla Fiorentini, Alessandro Vergara, Filomena Sica, Ermenegilda Parrilli, Maria Luisa Tutino
The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborisation, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies...
October 24, 2017: Biotechnology Progress
Sabarinath Thankappan, Rajneesh Rana, Arun Thachappully Remesh, Valsala Rekha, Viswas Konasagara Nagaleekar, Bhavani Puvvala
AIM: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii. MATERIALS AND METHODS: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography...
September 2017: Veterinary World
J Seshu, Trever C Smith, Ying-Han Lin, S L Rajasekhar Karna, Christine L Miller, Tricia Van Laar
Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re...
2018: Methods in Molecular Biology
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
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
Huawei He, Shuguang Wei, Yejing Wang, Lina Liu, Zhenzhen Li, Peng Zhao, Huaipu Chang, Ping Zhao
Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Mehdi Imani, Hossein Zarei Jaliani, Mohammad Hassan Kheirandish, Mahnaz Azadpour
OBJECTIVES: A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification. MATERIALS AND METHODS: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+)...
April 2017: Iranian Journal of Basic Medical Sciences
Yuguang Wang, Qian Liu, Hongbo Zhou, Xinhua Chen
The cysteine desulfurase (SufS) gene of Sulfobacillus acidophilus TPY, a Gram-positive bacterium isolated from deep-sea hydrothermal vent, was cloned and over-expressed in E. coli BL21. The recombinant SufS protein was purified by one-step affinity chromatography. The TPY SufS contained a well conserved motif RXGHHCA as found in that of other microorganisms, suggesting that it belonged to group II of cysteine desulfurase family. The recombinant TPY SufS could catalyze the conversion of l-cysteine to l-alanine and produce persulfide, and the enzyme activity was 95 μ/μL of sulfur ion per minute...
December 2017: 3 Biotech
Valentina Kalichuk, Axelle Renodon-Cornière, Ghislaine Béhar, Federico Carrión, Gonzalo Obal, Mike Maillasson, Barbara Mouratou, Véronique Préat, Frédéric Pecorari
Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d...
October 4, 2017: Biotechnology and Bioengineering
Jian Zhang, Aiyou Sun, Yuguo Dong, Dongzhi Wei
Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system...
October 2, 2017: Applied Biochemistry and Biotechnology
Ramona Schlesinger, Anneliese Cousin, Joachim Granzin, Renu Batra-Safferling
Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column...
2017: Methods in Cell Biology
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