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Molecular Biotechnology

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https://www.readbyqxmd.com/read/29230626/efficient-one-step-fusion-pcr-based-on-dual-asymmetric-primers-and-two-step-annealing
#1
Yilan Liu, Jinjin Chen, Anders Thygesen
Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized...
December 11, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29214500/enhancing-cellulase-and-hemicellulase-production-in-trichoderma-orientalis-eu7-22-via-knockout-of-the-crea
#2
Chuannan Long, Yijin Cheng, Jingjing Cui, Jian Liu, Lihui Gan, Bin Zeng, Minnan Long
The role of the transcription factor creA-mediating carbon catabolite repression in Trichoderma orientalis EU7-22 was investigated for cellulase and hemicellulase production. The binary vector pUR5750G/creA::hph was constructed to knock out creA by homologous integration, generating the ΔcreA mutant Trichoderma orientalis CF1D. For strain CF1D, the filter paper activities (FPA), endoglucanase activities (CMC), cellobiohydrolase activity(CBH), β-glucosidase activity (BG), xylanase activity (XYN), and extracellular protein concentration were 1...
December 6, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29214499/microbial-degradation-of-forensic-samples-of-biological-origin-potential-threat-to-human-dna-typing
#3
REVIEW
Hirak Ranjan Dash, Surajit Das
Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials...
December 6, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29214498/cloning-and-aggregation-characterization-of-rubber-elongation-factor-and-small-rubber-particle-protein-from-ficus-carica
#4
Saki Yokota, Yurina Suzuki, Keisuke Saitoh, Sakihito Kitajima, Norimasa Ohya, Takeshi Gotoh
Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP...
December 6, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29197992/identification-of-reference-genes-for-analysis-of-microrna-expression-patterns-in-equine-chorioallantoic-membrane-and-serum
#5
Pouya Dini, Shavahn C Loux, Kirsten E Scoggin, Alejandro Esteller-Vico, Edward L Squires, Mats H T Troedsson, Peter Daels, Barry A Ball
MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy...
December 2, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29196986/over-expression-of-atpap1-transcriptional-factor-enhances-phenolic-acid-production-in-transgenic-roots-of-leonurus-sibiricus-l-and-their-biological-activities
#6
Przemysław Sitarek, Tomasz Kowalczyk, Patricia Rijo, Adam J Białas, Marzena Wielanek, Halina Wysokińska, Catarina Garcia, Monika Toma, Tomasz Śliwiński, Ewa Skała
This study examines the production of five phenolic acids (chlorogenic acid, neochlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) following over-expression of AtPAP1 transcription factor by four transgenic root clones of Leonurus sibiricus after Agrobacterium rhizogenes transformation. The AtPAP1 expression level was estimated by quantitative real-time PCR. High levels of phenolic acids were found in the transgenic roots of L. sibiricus and were determined by high-performance liquid chromatography-mass spectrometry analysis...
December 1, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29196985/virus-induced-gene-silencing-vigs-in-aegilops-tauschii-and-its-use-in-functional-analysis-of-aetdreb2
#7
Elahe Tavakol
Among the available reverse genetic approaches for studying gene function, virus-induced gene silencing (VIGS) has several advantages. It allows rapid characterization of gene function independent of stable transformation, which is basically difficult to achieve in monocots, and offers the potential to silence individual or multiple genes of a gene family. In order to establish a VIGS system in Aegilops tauschii, modified vectors derived from Barley stripe mosaic virus (BSMV) were used for silencing a phytoene desaturase gene that provides a convenient visual reporter for silencing...
December 1, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29192396/insights-into-the-synergistic-biodegradation-of-waste-papers-using-a-combination-of-thermostable-endoglucanase-and-cellobiohydrolase-from-chaetomium-thermophilum
#8
Weiguang Li, Peng Ji, Qinzheng Zhou, Chengyao Hua, Chao Han
Enzymatic hydrolysis is considered an efficient and environmental strategy for the degradation of organic waste materials. Compared to mesophilic cellulases, thermostable cellulases with considerable activity are more advantageous in waste paper hydrolysis, particularly in terms of their participation in synergistic action. In this study, the synergistic effect of two different types of thermostable Chaetomium thermophilum cellulases, the endoglucanase CTendo45 and the cellobiohydrolase CtCel6, on five common kinds of waste papers was investigated...
November 30, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29143175/vectors-for-expression-of-signal-peptide-dependent-proteins-in-baculovirus-insect-cell-systems-and-their-application-to-expression-and-purification-of-the-high-affinity-immunoglobulin-gamma-fc-receptor-i-in-complex-with-its-gamma-chain
#9
Le T M Le, Jens R Nyengaard, Monika M Golas, Bjoern Sander
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER)...
November 15, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29138983/cloning-expression-analysis-and-enzyme-activity-assays-of-the-%C3%AE-carbonic-anhydrase-gene-from-chlamydomonas-sp-ice-l
#10
Changfeng Qu, Yingying He, Zhou Zheng, Meiling An, Lulu Li, Xixi Wang, Xiaodong He, Yibin Wang, Fangming Liu, Jinlai Miao
The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0...
November 14, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29128956/cell-surface-display-of-merr-on-saccharomyces-cerevisiae-for-biosorption-of-mercury
#11
Qinguo Wei, Jiakuo Yan, Yao Chen, Lei Zhang, Xiaoyang Wu, Shuai Shang, Shisheng Ma, Tian Xia, Shuyu Xue, Honghai Zhang
The metalloregulatory protein MerR which plays important roles in mer operon system exhibits high affinity and selectivity toward mercury (II) (Hg(2+)). In order to improve the adsorption ability of Saccharomyces cerevisiae for Hg(2+), MerR was displayed on the surface of S. cerevisiae for the first time with an α-agglutinin-based display system in this study. The merR gene was synthesized after being optimized and added restriction endonuclease sites EcoR I and Mlu I. The display of MerR was indirectly confirmed by the enhanced adsorption ability of S...
November 11, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29058211/the-effects-of-one-amino-acid-substitutions-at-the-c-terminal-region-of-thermostable-l2-lipase-by-computational-and-experimental-approach
#12
Hartini Ahmad Sani, Fairolniza Mohd Shariff, Raja Noor Zaliha Raja Abd Rahman, Thean Chor Leow, Abu Bakar Salleh
The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2...
October 20, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28993982/comparative-assessment-on-the-expression-level-of-recombinant-human-follicle-stimulating-hormone-fsh-in-serum-containing-versus-protein-free-culture-media
#13
Seyedeh Hoda Jazayeri, Amir Amiri-Yekta, Hamid Gourabi, Baharak Abd Emami, Zahra Halfinezhad, Somayeh Abolghasemi, Nayeralsadat Fatemi, Abbas Daneshipour, Mohammad Hossein Ghahremani, Mohammad Hossein Sanati, Mohammad Reza Khorramizadeh
Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28965203/assessment-of-carrot-callus-as-biofactories-of-an-atherosclerosis-oral-vaccine-prototype
#14
Dania O Govea-Alonso, Marlene A Tello-Olea, Josué Beltrán-López, Elizabeth Monreal-Escalante, Jorge A Salazar-Gonzalez, Bernardo Bañuelos-Hernández, Sergio Rosales-Mendoza
Atherosclerosis is a pathology leading to cardiovascular diseases with high epidemiologic impact; thus, new therapies are required to fight this global health issue. Immunotherapy is a feasible approach to treat atherosclerosis and given that genetically engineered plants are attractive hosts for vaccine development; we previously proved that the plant cell is able to synthesize a chimeric protein called CTB:p210:CETPe, which is composed of the cholera toxin B subunit (CTB) as immunogenic carrier and target epitopes from the cholesteryl ester transfer protein (CETP461-476) and apolipoprotein B100 (p210)...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28921459/chimeric-virus-as-a-source-of-the-potato-leafroll-virus-antigen
#15
Eugene V Skurat, Konstantin O Butenko, Olga A Kondakova, Nikolai A Nikitin, Olga V Karpova, Yuri F Drygin, Joseph G Atabekov
Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28884294/a-concise-review-of-microrna-exploring-the-insights-of-microrna-regulations-in-bacterial-viral-and-metabolic-diseases
#16
REVIEW
Ahsan Naveed, Sajjad Ur-Rahman, Sabahat Abdullah, Muhammad Ammar Naveed
MicroRNA (miRNA) is a small section of ribonucleic acid (RNA) that reduces the protein formation by making the pair of the complementary piece of mRNA. The genes of miRNA are present as transcriptional or polycistronic units in the chromosomes. The cellular multiplication, separation and existence like the multitude of genetic functions are affected by miRNA. Nearly 50% of identified miRNA are located in the residence in the intronic part of the genes. The mature miRNA is yielded in two steps. Drosha and RNA-induced silencing complex are the catalysts that play an important role in miRNA synthesis...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28828714/genetic-engineering-a-possible-strategy-for-protein-energy-malnutrition-regulation
#17
REVIEW
Praveen Guleria, Vineet Kumar, Shiwani Guleria
Protein-energy malnutrition (PEM) has adversely affected the generations of developing countries. It is a syndrome that in severity causes death. PEM generally affects infants of 1-5 age group. This manifestation is maintained till adulthood in the form of poor brain and body development. The developing nations are continuously making an effort to curb PEM. However, it is still a prime concern as it was in its early years of occurrence. Transgenic crops with high protein and enhanced nutrient content have been successfully developed...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28801881/transgenic-analysis-reveals-5-abbreviated-osrglp2-promoter-s-as-responsive-to-abiotic-stresses
#18
Shahzad Hussain Shah, Azka Noureen, Farah Deeba, Tasawar Sultana, Stefanie Dukowic-Schulze, Changbin Chen, Syed Muhammad Saqlan Naqvi
Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5' deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses...
December 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28801830/cloning-and-expression-analysis-of-human-amelogenin-in-nicotiana-benthamiana-plants-by-means-of-a-transient-expression-system
#19
Mattia Pegoraro, Slavica Matić, Barbara Pergolizzi, Luca Iannarelli, Andrea M Rossi, Marco Morra, Emanuela Noris
Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons...
October 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28801725/optimization-of-soluble-expression-and-purification-of-recombinant-human-rhinovirus-type-14-3c-protease-using-statistically-designed-experiments-isolation-and-characterization-of-the-enzyme
#20
Georgia Antoniou, Irineos Papakyriacou, Christos Papaneophytou
Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5...
October 2017: Molecular Biotechnology
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