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Molecular Biotechnology

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https://www.readbyqxmd.com/read/28631058/expression-and-characterization-of-glucose-oxidase-from-aspergillus-niger-in-yarrowia-lipolytica
#1
Fatemeh Khadivi Derakshan, Farshad Darvishi, Mehrouz Dezfulian, Catherine Madzak
Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y...
June 19, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28597202/erratum-to-design-of-a-lentiviral-vector-for-the-inducible-expression-of-myc-a-new-strategy-for-construction-approach
#2
Onur Tokgun, Francesco Paolo Fiorentino, Pervin Elvan Tokgun, Jun Yokota, Hakan Akca
No abstract text is available yet for this article.
June 8, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28585118/analysis-of-promoters-of-arabidopsis-thaliana-divergent-gene-pair-serat3-2-and-idh-iii-shows-serat3-2-promoter-is-nested-within-the-idh-iii-promoter
#3
Ritesh Kumar Raipuria, Vajinder Kumar, Kadur Narayan Guruprasad, Shripad Ramachandra Bhat
Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp...
June 5, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28580552/ability-of-t1-lipase-to-degrade-amorphous-p-3hb-structural-and-functional-study
#4
Rauda A Mohamed, Abu Bakar Salleh, Adam Thean Chor Leow, Normi M Yahaya, Mohd Basyaruddin Abdul Rahman
An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53...
June 3, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28573450/cloning-production-and-characterization-of-a-glycoside-hydrolase-family-7-enzyme-from-the-gut-microbiota-of-the-termite-coptotermes-curvignathus
#5
James Sy-Keen Woon, Patricia Jie Hung King, Mukram Mohamed Mackeen, Nor Muhammad Mahadi, Wan Mohd Khairulikhsan Wan Seman, William J Broughton, Abdul Munir Abdul Murad, Farah Diba Abu Bakar
Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris...
June 1, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28573449/medium-optimization-for-improved-production-of-dihydrolipohyl-dehydrogenase-from-bacillus-sphaericus-pad-91-in-escherichia-coli
#6
Hamid Shahbazmohammadi, Eskandar Omidinia
Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM)...
June 1, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28567687/integrated-method-for-purification-and-single-particle-characterization-of-lentiviral-vector-systems-by-size-exclusion-chromatography-and-tunable-resistive-pulse-sensing
#7
Susanne Heider, Julien Muzard, Marianne Zaruba, Christoph Metzner
Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes...
May 31, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28567686/tobacco-rattle-virus-based-silencing-of-enoyl-coa-reductase-gene-and-its-role-in-resistance-against-cotton-wilt-disease
#8
Roma Mustafa, Muhammad Hamza, Hira Kamal, Shahid Mansoor, Jodi Scheffler, Imran Amin
A Tobacco rattle virus (TRV)-based virus-induced gene silencing assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of the Enoyl-CoA reductase (GhECR) gene in pathogen defense. Amino acid sequence alignment of Arabidopsis ECR with homologous sequence from G. hirsutum, G. arboreum, G. herbaceum and G. barbadense showed that ECRs are highly conserved among these species. TRV-based silencing of GhECR gene in G...
May 31, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28526937/molecular-confirmation-of-intraspecific-tomato-solanum-lycopersicum-hybrids-and-their-evaluation-against-late-blight-and-cucumber-mosaic-virus
#9
Amjad Hameed, Muhammad Yussouf Saleem, Khalid Pervaiz Akhtar, Muhammad Shoaib, Qumer Iqbal, Muhammad Asghar
Tomato is one of the most consumed vegetables in the world. Diseases are the number one concern in the development of high-yield and disease-resistant tomato hybrids which is the foremost priority of breeders. Present study was conducted (1) to develop DNA-based markers for genetic confirmation of tomato F1 hybrids, (2) to utilize sequenced characterized amplified region (SCAR) marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato and (3) to evaluate male and female parental genotypes and their F1 hybrids against late blight (LB) and cucumber mosaic virus (CMV)...
May 19, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28509990/transient-activation-of-reprogramming-transcription-factors-using-protein-transduction-facilitates-conversion-of-human-fibroblasts-toward-cardiomyocyte-like-cells
#10
Zaniar Ghazizadeh, Hassan Rassouli, Hananeh Fonoudi, Mehdi Alikhani, Mahmood Talkhabi, Amir Darbandi-Azar, Shuibing Chen, Hossein Baharvand, Nasser Aghdami, Ghasem Hosseini Salekdeh
Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine)...
May 16, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28500482/improved-yield-of-high-molecular-weight-hyaluronic-acid-production-in-a-stable-strain-of-streptococcus-zooepidemicus-via-the-elimination-of-the-hyaluronidase-encoding-gene
#11
Navid Pourzardosht, Mohammad Javad Rasaee
Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana(r)-R as a recombinant suicide plasmid...
May 12, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28484957/characterization-of-recombinant-thermococcus-kodakaraensis-kod-dna-polymerases-produced-using-silkworm-baculovirus-expression-vector-system
#12
Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee
The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively...
May 8, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28421327/metal-dna-interactions-improve-signal-in-high-resolution-melting-of-dna-for-species-differentiation-of-plasmodium-parasite
#13
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
#14
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/28447263/design-of-a-lentiviral-vector-for-the-inducible-expression-of-myc-a-new-strategy-for-construction-approach
#15
Onur Tokgun, Francesco Paolo Fiorentino, Pervin Elvan Tokgun, Jun Yokota, Hakan Akca
Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345...
June 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
#16
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
#17
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
#18
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
#19
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
#20
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
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