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Molecular Biotechnology

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https://www.readbyqxmd.com/read/28421327/metal-dna-interactions-improve-signal-in-high-resolution-melting-of-dna-for-species-differentiation-of-plasmodium-parasite
#1
Priyamvada Jain, Babina Chakma, Naveen Singh, Sanjukta Patra, Pranab Goswami
The success of high-resolution melting (HRM) analysis for distinguishing similar DNAs with minor base mismatch differences is limited. Here, metal-mediated structural change in DNA has been exploited to amplify HRM signals leading to differentiation of target DNAs in an orthologous gene corresponding to four Plasmodium species. Conserved 26-mer ssDNAs from ldh gene of the four Plasmodium species were employed as targets. A capture probe (CP) that is fully complementary to the Plasmodium falciparum target (FT) and has two base mismatches each, with the targets of Plasmodium vivax (VT), Plasmodium malariae, (MT), and Plasmodium ovale (OT), was considered...
April 18, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28374116/designing-an-escherichia-coli-strain-for-phenylalanine-overproduction-by-metabolic-engineering
#2
Neetu Tyagi, Deepti Saini, Richa Guleria, Krishna Jyoti Mukherjee
The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG (fbr) performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications...
April 3, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28349302/a-mutant-sumo-facilitates-quick-plasmid-construction-for-expressing-proteins-with-native-n-termini-after-tag-removal
#3
Yuzhu Zhang, Yuting Fan
Sumo is one of the fusion tags commonly used to enhance the expression and the solubility of recombinant proteins. One advantage of using sumo is that the removal of the sumo tag is highly specific because its recognition by a sumo protease is determined by its structural characteristics, instead of the sequence of a short peptide. Recently, it was reported that sumo could also be used as a protease recognition site to facilitate the removal of other fusion tags, such as MBP, when sumo itself is not suitable to enhance the solubility of a particular target protein...
March 27, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28342150/expression-and-characterization-of-human-%C3%AE-1-4-galactosyltransferase-1-%C3%AE-4galt1-using-silkworm-baculovirus-expression-system
#4
Daisuke Morokuma, Jian Xu, Masato Hino, Hiroaki Mon, Jasmeen S Merzaban, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee
Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm-BEVS...
March 24, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28342149/identification-and-preparation-of-a-novel-chemokine-receptor-binding-domain-in-the-cytoplasmic-regulator-frount
#5
Akihiro Sonoda, Sosuke Yoshinaga, Kaori Yunoki, Soichiro Ezaki, Kotaro Yano, Mitsuhiro Takeda, Etsuko Toda, Yuya Terashima, Kouji Matsushima, Hiroaki Terasawa
FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors...
March 24, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28332030/molecular-cloning-expression-and-characterization-of-pectin-methylesterase-ctpme-from-clostridium-thermocellum
#6
Vikky Rajulapati, Arun Goyal
Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells...
March 22, 2017: Molecular Biotechnology
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
#7
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/28271340/an-overview-on-the-enhancement-of-enantioselectivity-and-stability-of-microbial-epoxide-hydrolases
#8
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
#9
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
#10
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
#11
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
#12
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/28025776/expression-polyubiquitination-and-therapeutic-potential-of-recombinant-e6e7-from-hpv16-antigens-fused-to-ubiquitin
#13
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
#14
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/28012062/a-synthetic-hybrid-promoter-for-xylose-regulated-control-of-gene-expression-in-saccharomyces-yeasts
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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