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Purification his tagged protein

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https://www.readbyqxmd.com/read/28730304/novel-ribonuclease-activity-of-cusativin-from-cucumis-sativus-for-mapping-nucleoside-modifications-in-rna
#1
Balasubrahmanyam Addepalli, Sarah Venus, Priti Thakur, Patrick A Limbach
A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein...
July 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28706572/a-microwell-printing-fabrication-strategy-for-the-on-chip-templated-biosynthesis-of-protein-microarrays-for-surface-plasmon-resonance-imaging
#2
Gerald Manuel, Andrej Lupták, Robert M Corn
A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein...
September 22, 2016: Journal of Physical Chemistry. C, Nanomaterials and Interfaces
https://www.readbyqxmd.com/read/28698826/expression-refolding-and-spectroscopic-characterization-of-fibronectin-type-iii-fniii-homology-domains-derived-from-human-fibronectin-leucine-rich-transmembrane-protein-flrt-1-2-and-3
#3
Lila Yang, Maria Hansen Falkesgaard, Peter Waaben Thulstrup, Peter Schledermann Walmod, Leila Lo Leggio, Kim Krighaar Rasmussen
The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans-interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis-interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation...
2017: PeerJ
https://www.readbyqxmd.com/read/28685146/his-flag-tag-as-a-fusion-partner-of-glycosylated-human-interferon-gamma-and-its-mutant-gain-or-loss
#4
Elena Krachmarova, Milena Tileva, Elena Lilkova, Peicho Petkov, Klaus Maskos, Nevena Ilieva, Ivan Ivanov, Leandar Litov, Genoveva Nacheva
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28670287/preparative-sds-page-as-an-alternative-to-his-tag-purification-of-recombinant-amelogenin
#5
Claire M Gabe, Steven J Brookes, Jennifer Kirkham
Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28668496/generation-of-nanobodies-against-slyd-and-development-of-tools-to-eliminate-this-bacterial-contaminant-from-recombinant-proteins
#6
Yaozhong Hu, Ema Romão, Didier Vertommen, Cécile Vincke, Francisco Morales-Yánez, Carlos Gutiérrez, Changxiao Liu, Serge Muyldermans
The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response...
June 28, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28661426/rational-design-of-recombinant-papain-like-cysteine-protease-optimal-domain-structure-and-expression-conditions-for-wheat-derived-enzyme-triticain-%C3%AE
#7
Neonila V Gorokhovets, Vladimir A Makarov, Anastasiia I Petushkova, Olga S Prokopets, Mikhail A Rubtsov, Lyudmila V Savvateeva, Evgeni Yu Zernii, Andrey A Zamyatnin
Triticain-α is a papain-like cysteine protease from wheat (Triticumaestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones...
June 29, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28628836/an-affinity-based-aqueous-two-phase-mixed-micellar-system-and-its-purification-of-yeast-3-5-bisphosphate-nucleotidase
#8
Yao Liu, Mei-Hao Sun, Shi-Kuan Shao, Gang Deng
Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system...
May 30, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28625912/isolation-of-recombinant-human-untagged-glyceraldehyde-3-phosphate-dehydrogenase-from-e-%C3%A2-coli-producer-strain
#9
K V Barinova, M A Eldarov, E V Khomyakova, V I Muronetz, E V Schmalhausen
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass)...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624997/expression-and-purification-of-cytochrome-p450-55b1-from-chlamydomonas-reinhardtii-and-its-application-in-nitric-oxide-biosensing
#10
Bin Gong, Xiaosheng Liang, Yong Li, Qian Xiao, Panchun Yang, Yunhua Wu
Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0...
June 17, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28624493/expression-of-the-c-terminal-domain-of-human-apolipoprotein-a-i-using-a-chimeric-apolipoprotein
#11
Daniel E Sallee, James V C Horn, Lukas A Fuentes, Paul M M Weers
Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28575696/regiospecific-radiolabelling-of-nanofitin-on-ni-magnetic-beads-with-18-f-fbem-and-in-vivo-pet-studies
#12
Sylvestre Dammicco, Marine Goux, Christian Lemaire, Guillaume Becker, Mohamed Ali Bahri, Alain Plenevaux, Mathieu Cinier, André Luxen
INTRODUCTION: Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs...
April 27, 2017: Nuclear Medicine and Biology
https://www.readbyqxmd.com/read/28569168/construct-design-production-and-characterization-of-plasmodium-falciparum-48-45-r0-6c-subunit-protein-produced-in-lactococcus-lactis-as-candidate-vaccine
#13
Susheel K Singh, Will Roeffen, Ulrik H Mistarz, Bishwanath Kumar Chourasia, Fen Yang, Kasper D Rand, Robert W Sauerwein, Michael Theisen
BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies...
May 31, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28517015/genetic-incorporation-of-4-fluorohistidine-into-peptides-enables-selective-affinity-purification
#14
Christine M Ring, Emil S Iqbal, David E Hacker, Matthew C T Hartman, T Ashton Cropp
Due to the lowered pKa of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.
May 31, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28487257/functional-efficacy-of-human-recombinant-fgf-2s-tagged-with-his-6-and-his-asn-6-at-the-n-and-c-termini-in-human-gingival-fibroblast-and-periodontal-ligament-derived-cells
#15
Ji-Hye Lee, Ji-Eun Lee, Kyung-Jung Kang, Young-Joo Jang
Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein...
May 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28470597/harnessing-the-profinity-exact%C3%A2-system-for-expression-and-purification-of-heterologous-proteins-in-e-coli
#16
Yoav Peleg, Vadivel Prabahar, Dominika Bednarczyk, Tamar Unger
Highly purified recombinant proteins in large quantities are valuable material for biochemical and structural studies. To achieve this goal, versatile tools were developed to increase the expression of the recombinant proteins and to facilitate the purification process. Fusion tags are commonly used for enhancing expression and solubility and some can be used in the purification process. However, these tags may need to be removed by treatment with specific proteases in order to obtain the tag-free protein. The Profinity eXact™ system provides an alternative system for a fusion tag, enhancing expression and purification in one-step...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28438686/expression-purification-and-molecular-characterization-of-a-novel-endoglucanase-protein-from-bacillus-subtilis-sb13
#17
Xuefang Guan, Penglian Chen, Qingxian Xu, Lei Qian, Juqing Huang, Bin Lin
Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and β-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain...
June 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28415641/identification-and-screening-of-effective-protective-antigens-for-channel-catfish-against-streptococcus-iniae
#18
Yajun Wang, Erlong Wang, Yang He, Kaiyu Wang, Qian Yang, Jun Wang, Yi Geng, Defang Chen, Xiaoli Huang, Ping Ouyang, Weimin Lai, Cunbin Shi
Vaccination is a potential approach for prevention and control of disease in fish. The use of genetically engineered vaccines is an effective method and a green intervention to control bacterial infection in aquaculture. However, efforts to develop these vaccines are limited by the lack of conserved protective antigens. In this study, three candidate immunogens (Srr, NeuA, and Hsp) of the pathogenic Streptococcus iniae strain DGX07 isolated from diseased channel catfish were identified and analyzed. Molecular cloning, expression, and purification of candidate antigen genes were carried out to obtain the candidate immunogens in the form of recombinant subunit vaccines...
May 9, 2017: Oncotarget
https://www.readbyqxmd.com/read/28330196/cloning-and-expression-of-saccharomyces-cerevisiae-suc2-gene-in-yeast-platform-and-characterization-of-recombinant-enzyme-biochemical-properties
#19
Nooshin Mohandesi, Seyed Omid Ranaei Siadat, Kamahldin Haghbeen, Ardeshir Hesampour
Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28330118/cloning-expression-purification-and-characterization-of-lipase-from-bacillus-licheniformis-isolated-from-hot-spring-of-himachal-pradesh-india
#20
Gagandeep Kaur, Amninder Singh, Rohit Sharma, Vinay Sharma, Swati Verma, Pushpender K Sharma
In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg(-1) and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i...
June 2016: 3 Biotech
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