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Protein expression and purification

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https://www.readbyqxmd.com/read/29345069/automation-aided-optimization-of-cloning-expression-and-purification-of-enzymes-of-the-bacterial-sialic-acid-catabolic-and-sialylation-pathways-enzymes-for-structural-studies
#1
Sneha Bairy, Lakshmi Narayanan Gopalan, Thanuja Gangi Setty, Sathya Srinivasachari, Lavanyaa Manjunath, Jay Prakash Kumar, Sai R Guntupalli, Sucharita Bose, Vinod Nayak, Swagatha Ghosh, Nitish Sathyanarayanan, Rhawnie Caing-Carlsson, Weixiao Yuan Wahlgren, Rosmarie Friemann, S Ramaswamy, Muniasamy Neerathilingam
The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway...
January 17, 2018: Microbial Biotechnology
https://www.readbyqxmd.com/read/29344087/determination-of-the-native-features-of-the-exoglucanase-cel48s-from-clostridium-thermocellum
#2
Ya-Jun Liu, Shiyue Liu, Sheng Dong, Renmin Li, Yingang Feng, Qiu Cui
Background: Clostridium thermocellum is considered one of the most efficient natural cellulose degraders because of its cellulosomal system. As the major exoglucanase of cellulosome in C. thermocellum, Cel48S plays key roles and influences the activity and features of cellulosome to a great extent. Thus, it is of great importance to reveal the enzymatic features of Cel48S. However, Cel48S has not been well performed due to difficulties in purifying either recombinant or native Cel48S proteins...
2018: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/29339216/in-vivo-biotinylated-calpastatin-improves-the-affinity-purification-of-human-m-calpain
#3
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
https://www.readbyqxmd.com/read/29337983/wor1-establishes-opaque-cell-fate-through-inhibition-of-the-general-co-repressor-tup1-in-candida-albicans
#4
Selma S Alkafeef, Clinton Yu, Lan Huang, Haoping Liu
The pathogenic fungus Candida albicans can undergo phenotypic switching between two heritable states: white and opaque. This phenotypic plasticity facilitates its colonization in distinct host niches. The master regulator WOR1 is exclusively expressed in opaque phase cells. Positive feedback regulation by Wor1 on the WOR1 promoter is essential for opaque formation, however the underlying mechanism of how Wor1 functions is not clear. Here, we use tandem affinity purification coupled with mass spectrometry to identify Wor1-interacting proteins...
January 16, 2018: PLoS Genetics
https://www.readbyqxmd.com/read/29331014/biochemical-characterization-of-recombinant-thermostable-cohnella-sp-a01-%C3%AE-glucanase
#5
Meysam Rezaie, Saeed Aminzadeh, Farid Heidari, Masoud Mashhadi Akbar Boojar, Ali Asghar Karkhane
Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are release by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3)...
January 13, 2018: Iranian Biomedical Journal
https://www.readbyqxmd.com/read/29329805/preparative-expression-and-purification-of-a-nacreous-protein-n16-and-testing-its-effect-on-osteoporosis-rat-model
#6
Zhen-Yan Xu, Yu-Ling Liu, Jia-Bi Lin, Ke-Ling Cheng, Yong-Gang Wang, Hong-Liang Yao, Wei-Peng, Hoi-Yan Wu, Wei-Wei Su, Pang-Chui Shaw, Pei-Bo Li
N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized...
January 10, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29323419/evaluation-of-viral-contamination-in-a-baculovirus-expression-system
#7
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
https://www.readbyqxmd.com/read/29322424/expression-and-purification-of-jak1-and-socs1-for-structural-and-biochemical-studies
#8
Nicholas P D Liau, Jeffrey J Babon
Interferon gamma (IFNγ) is a potent inflammatory and immune cytokine. IFNγ signals via the interferon gamma receptor (IFNGR), which is constitutively bound to Janus Kinase (JAK) 1 and JAK2 via its intracellular domain. These two JAK proteins then initiate the inflammatory signaling cascade. The most potent inhibitor of IFNγ signaling is Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 negatively regulates IFNγ signaling pathway (and other pathways) by directly inhibiting JAKs. Here, we describe a protocol for the recombinant production and purification of the JAK1 kinase domain and its inhibitor SOCS1, for structural and biochemical studies...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29321325/the-identification-and-characterization-of-sindbis-virus-rna-host-protein-interactions
#9
Autumn T LaPointe, Natasha N Gebhart, Megan E Meller, Richard W Hardy, Kevin J Sokoloski
Arthropod-borne viruses, such as the members of genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29318540/identification-of-proteolytic-cleavage-sites-of-epha2-by%C3%A2-membrane-type-1-matrix-metalloproteinase-on-the%C3%A2-surface-of-cancer-cells
#10
Keiji Kikuchi, Hiroko Kozuka-Hata, Masaaki Oyama, Motoharu Seiki, Naohiko Koshikawa
Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification ...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29318516/cloning-and-expression-of-h-influenzae-49247-iga-protease-in-e-coli
#11
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
https://www.readbyqxmd.com/read/29317682/piperonylic-acid-stimulates-keratinocyte-growth-and-survival-by-activating-epidermal-growth-factor-receptor-egfr
#12
Dohyun Lee, Jinsun Lim, Kyung-Chul Woo, Kyong-Tai Kim
Epidermal growth factor (EGF) stimulates cell growth, proliferation, and survival. The biological benefits of EGF have been utilized in medical uses for improving wound healing as well as in today's skin cosmetics. EGF has been found in urine, saliva, milk, and plasma, but its efficient isolation remains a difficult task. With technical advances, recombinant protein purification technique has been used for EGF production. However, the recombinant EGF is still expensive and keeping it with stable activity is difficult to be used widely...
January 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29314780/hpv-16-targeted-dna-vaccine-expression-the-role-of-purification
#13
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
https://www.readbyqxmd.com/read/29313422/selection-purification-and-characterization-of-a-her2-targeting-soluble-designed-ankyrin-repeat-protein-by-e-coli-surface-display-using-her2-positive-melanoma-cells
#14
Xiaofei Chen, Xiaoxiao Yu, Xiaoda Song, Li Liu, Yuting Yi, Wenbing Yao, Xiangdong Gao
Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells...
January 9, 2018: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/29310786/production-and-purification-of-recombinant-human-sparc
#15
Gail Workman, Amy D Bradshaw
The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta...
2018: Methods in Cell Biology
https://www.readbyqxmd.com/read/29310782/the-biochemistry-and-immunohistochemistry-of-versican
#16
Stephen P Evanko, Christina K Chan, Pamela Y Johnson, Charles W Frevert, Thomas N Wight
Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multimolecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation...
2018: Methods in Cell Biology
https://www.readbyqxmd.com/read/29310781/expression-and-purification-of-recombinant-fibulins-in-mammalian-cells
#17
Katsuhiro Hanada, Takako Sasaki
Functional studies of extracellular proteins are often performed using coimmunoprecipitation without purified proteins. However, in order to exclude unspecific reactions of contaminants and for quantitative analysis of specific functions, it is necessary to use purified proteins. It is usually very difficult, however, to purify sufficient amounts of reasonably pure extracellular matrix proteins from tissue samples, but the recombinant expression and purification of proteins in eukaryotic expression systems including insect cells and mammalian cells has proven an alternative powerful method...
2018: Methods in Cell Biology
https://www.readbyqxmd.com/read/29306432/methods-for-the-detection-study-and-dynamic-profiling-of-o-glcnac-glycosylation
#18
John W Thompson, Matthew E Griffin, Linda C Hsieh-Wilson
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease...
2018: Methods in Enzymology
https://www.readbyqxmd.com/read/29305897/cloning-expression-and-purification-of-virb10-protein-of-brucella-melitensis-and-to-evaluate-its-role-as-a-serological-marker-for-brucella-infection-in-experimental-and-natural-hosts
#19
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
https://www.readbyqxmd.com/read/29304331/rapid-and-scalable-characterization-of-crispr-technologies-using-an-e-%C3%A2-coli-cell-free-transcription-translation-system
#20
Ryan Marshall, Colin S Maxwell, Scott P Collins, Thomas Jacobsen, Michelle L Luo, Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Yonghua He, Chase L Beisel, Vincent Noireaux
CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells...
January 4, 2018: Molecular Cell
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