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Molecular Biotechnology

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https://www.readbyqxmd.com/read/28324209/molecular-cloning-structural-modeling-and-the-production-of-soluble-triple-mutated-diphtheria-toxoid-k51e-g52e-e148k-co-expressed-with-molecular-chaperones-in-recombinant-escherichia-coli
#1
Naphatsamon Uthailak, Pornpimol Mahamad, Pamorn Chittavanich, Somchai Yanarojana, Wassana Wijagkanalan, Jean Petre, Watanalai Panbangred
CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97...
March 21, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28025776/expression-polyubiquitination-and-therapeutic-potential-of-recombinant-e6e7-from-hpv16-antigens-fused-to-ubiquitin
#2
Liliane M Fernandes de Oliveira, Mirian G Morale, Agtha A M Chaves, Marilene Demasi, Paulo L Ho
Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8(+) T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies...
December 26, 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/28013401/prolonged-production-and-aggregation-complexity-of-cold-active-lipase-from-pseudomonas-proteolytica-gbpi_hb61-isolated-from-cold-desert-himalaya
#3
Rahul Jain, Anita Pandey, Mukesh Pasupuleti, Veena Pande
Pseudomonas, being the common inhabitant of colder environments, are suitable for the production of cold-active enzymes. In the present study, a newly isolated strain of Pseudomonas from cold desert site in Indian Himalayan Region, was investigated for the production of cold-active lipase. The bacteria were identified as Pseudomonas proteolytica by 16S rDNA sequencing. Lipase production by bacteria was confirmed by qualitative assay using tributyrin and rhodamine-B agar plate method. The bacterium produced maximum lipase at 25 °C followed by production at 15 °C while utilizing olive, corn, as well as soybean oil as substrate in lipase production broth...
December 24, 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/28271340/an-overview-on-the-enhancement-of-enantioselectivity-and-stability-of-microbial-epoxide-hydrolases
#4
REVIEW
Priya Saini, Dipti Sareen
Epoxide hydrolases (EHs; 3.3.2.x) catalyze the enantioselective ring opening of racemic epoxides to the corresponding enantiopure vicinal diols and remaining equivalent unreacted epoxides. These epoxides and diols are used for the synthesis of chiral drug intermediates. With an upsurge in the methods for identification of novel microbial EHs, a lot of EHs have been discovered and utilized for kinetic resolution of racemic epoxides. However, there is still a constraint on the account of limited EHs being successfully applied on the preparative scale for industrial biotransformations...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28197768/expression-of-an-acid-urease-with-urethanase-activity-in-e-coli-and-analysis-of-urease-gene
#5
REVIEW
Xiaofeng Liu, Qian Zhang, Nandi Zhou, Yaping Tian
Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28194691/rnai-mediated-simultaneous-resistance-against-three-rna-viruses-in-potato
#6
Amir Hameed, Muhammad Nouman Tahir, Shaheen Asad, Rakhshanda Bilal, Joyce Van Eck, Georg Jander, Shahid Mansoor
RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28138902/ectopic-expression-of-plant-rna-chaperone-offering-multiple-stress-tolerance-in-e-coli
#7
Bushra Jabeen, S M Saqlan Naqvi, Tariq Mahmood, Tasawar Sultana, Madiha Arif, Fariha Khan
Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28132389/an-improved-method-for-identifying-specific-dna-protein-binding-sites-in-vitro
#8
Liangyan Wang, Huizhi Lu, Yunguang Wang, Su Yang, Hong Xu, Kaiying Cheng, Ye Zhao, Bing Tian, Yuejin Hua
Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28012062/a-synthetic-hybrid-promoter-for-xylose-regulated-control-of-gene-expression-in-saccharomyces-yeasts
#9
Ronald E Hector, Jeffrey A Mertens
Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DNA-binding repressor (XylR) from Caulobacter crescentus was used to block transcription from synthetic promoters based on the constitutive Ashbya gossypii TEF promoter. The new hybrid promoters were repressed in the absence of xylose and showed up to a 25-fold increase in the presence of xylose...
December 23, 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27975151/retraction-note-to-a-report-on-the-internal-retreat-meeting-of-the-stem-cell-network-north-rhine-westphalia
#10
Evangelia Kontopoulou, Oumaima Stambouli, Sanjana Mathew
No abstract text is available yet for this article.
December 14, 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27798764/a-report-on-the-internal-retreat-meeting-of-the-stem-cell-network-north-rhine-westphalia
#11
REVIEW
Evangelia Kontopoulou, Oumaima Stambouli, Sanjana Mathew
No abstract text is available yet for this article.
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27771851/in-vitro-refolding-and-characterization-of-recombinant-laccase-cota-from-bacillus-pumilus-mk001-and-its-potential-for-phenolics-degradation
#12
Sandeep Kumar, Kavish Kr Jain, Shikha Rani, Kailash N Bhardwaj, Manisha Goel, Ramesh Chander Kuhad
Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27761701/application-of-a-loop-mediated-isothermal-amplification-assay-for-rapid-detection-of-cow-components-adulterated-in-buffalo-milk-meat
#13
Rajib Deb, Gyanendra Singh Sengar, Umesh Singh, Sushil Kumar, R R Alyethodi, Rani Alex, T V Raja, A K Das, B Prakash
Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27757798/amplicon-based-rna-interference-targeting-v2-gene-of-cotton-leaf-curl-kokhran-virus-burewala-strain-can-provide-resistance-in-transgenic-cotton-plants
#14
Aneela Yasmeen, Sarfraz Kiani, Afshan Butt, Abdul Qayyum Rao, Faheem Akram, Aftab Ahmad, Idrees Ahmad Nasir, Tayyab Husnain, Shahid Mansoor, Imran Amin, Shaheen Aftab, Muhammad Zubair, Muhammad Nouman Tahir, Sohail Akhtar, Jodi Scheffler, Brian Scheffler
The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27752959/inhibition-of-telomerase-activity-using-an-egfp-intron-splicing-system-encoding-multiple-rnai-sequences
#15
O Sakiragaoglu, A L Munn
To inhibit telomerase activity, a construct which contains artificial introns in the enhanced green fluorescent protein (EGFP) gene that encodes small hairpin RNA (shRNA) sequences that target human telomerase reverse transcriptase (hTERT) gene expression was designed and tested for its effect on lung cancer cell line. On intron splicing from the construct, intronic sequences were released and formed shRNA in the cells. After transfection of the construct, hTERT mRNA expression decreased by approximately 55 % in A549 cells...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27734193/effect-of-a-flexible-linker-on-recombinant-expression-of-cell-penetrating-peptide-fusion-proteins-and-their-translocation-into-fungal-cells
#16
Zifan Gong, Mackenzie T Walls, Alisha N Karley, Amy J Karlsson
Cell-penetrating peptides (CPPs) are a class of small peptides that are able to cross cell membranes via direct translocation or endocytosis. They have been widely used to deliver tethered bioactive molecules to cells, but recombinantly producing CPPs as fusions to protein cargo leads to low yields. We used Escherichia coli cells to recombinantly produce genetic fusions of NPFSD (derived from a yeast endocytosis signal) and pVEC (derived from a murine vascular endothelium cadherin) to the N-terminus of green fluorescent protein (GFP) with and without a flexible glycine-serine linker between the CPP and GFP...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27714589/expression-of-two-novel-%C3%AE-glucosidases-from-chaetomium-atrobrunneum-in-trichoderma-reesei-and-characterization-of-the-heterologous-protein-products
#17
Ana C Colabardini, Mari Valkonen, Anne Huuskonen, Matti Siika-Aho, Anu Koivula, Gustavo H Goldman, Markku Saloheimo
Two novel GH3 family thermostable β-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa β-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60 °C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27683255/an-approach-for-zika-virus-inhibition-using-homology-structure-of-the-envelope-protein
#18
Sandun Fernando, Teshan Fernando, Michal Stefanik, Ludek Eyer, Daniel Ruzek
To find an effective drug for Zika virus, it is important to understand how numerous proteins which are critical for the virus' structure and function interact with their counterparts. One approach to inhibiting the flavivirus is to deter its ability to bind onto glycoproteins; however, the crystal structures of envelope proteins of the ever-evolving viral strains that decipher glycosidic or drug-molecular interactions are not always available. To fill this gap, we are reporting a holistic, simulation-based approach to predict compounds that will inhibit ligand binding onto a structurally unresolved protein, in this case the Zika virus envelope protein (ZVEP), by developing a three-dimensional general structure and analyzing sites at which ligands and small drug-like molecules interact...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27670285/biochemical-characterization-and-low-resolution-saxs-molecular-envelope-of-gh1-%C3%AE-glycosidase-from-saccharophagus-degradans
#19
Hevila Brognaro, Vitor Medeiros Almeida, Evandro Ares de Araujo, Vasily Piyadov, Maria Auxiliadora Morim Santos, Sandro Roberto Marana, Igor Polikarpov
The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C...
December 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27830536/virus-like-particles-derived-from-hiv-1-for-delivery-of-nuclear-proteins-improvement-of-production-and-activity-by-protein-engineering
#20
Marc-André Robert, Viktoria Lytvyn, Francis Deforet, Rénald Gilbert, Bruno Gaillet
Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4)...
November 9, 2016: Molecular Biotechnology
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