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https://www.readbyqxmd.com/read/28747746/one-step-purification-and-immobilization-of-extracellularly-expressed-sortase-a-by-magnetic-particles-to-develop-a-robust-and-recyclable-biocatalyst
#1
Xinrui Zhao, Haofei Hong, Xiaozhong Cheng, Shaozhong Liu, Tao Deng, Zhongwu Guo, Zhimeng Wu
Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels...
July 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28747584/codon-optimization-enables-the-zeocin-resistance-marker-s-use-in-the-ascomycete-yeast-debaryomyces-occidentalis
#2
Yuu Utashima, Satoshi Yamashita, Toshi-Hide Arima, Kazuo Masaki
No abstract text is available yet for this article.
July 27, 2017: Journal of General and Applied Microbiology
https://www.readbyqxmd.com/read/28743658/high-level-expression-and-characterization-of-solvent-tolerant-lipase
#3
Yu Wang, Dan Luo, Yunshi Zhao, Shufang Tian, Wenpeng Deng, Chunhua Li, Lixin Ma
In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile...
July 22, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28739800/cell-free-production-of-a-functional-oligomeric-form-of-a-chlamydia-major-outer-membrane-protein-momp-for-vaccine-development
#4
Wei He, Martina Felderman, Angela C Evans, Jia Geng, David Homan, Feliza Bourguet, Nicholas O Fischer, Yuanpei Li, Kit S Lam, Aleksandr Noy, Li Xing, R Holland Cheng, Amy Rasley, Craig D Blanchette, Kurt Kamrud, Nathanial Wang, Heather Gouvis, Todd C Peterson, Bolyn Hubby, Matthew A Coleman
Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen...
July 24, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28731580/rewiring-the-glucose-transportation-and-central-metabolic-pathways-for-overproduction-of-n-acetylglucosamine-in-bacillus-subtilis
#5
Yang Gu, Jieying Deng, Yanfeng Liu, Jianghua Li, Hyun-Dong Shin, Guocheng Du, Jian Chen, Long Liu
N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. in a previous study, we constructed the recombinant Bacillus subtilis strain BSGN6-PxylA -glmS-pP43NMK-GNA1 (BN0-GNA1) for microbial production of GlcNAc by pathway design and modular optimization. Here, we further improved the production of GlcNAc by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) was blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit iiA YyzE), ypqE (encoding the PTS system transporter subunit iiA YpqE), and ptsG (encoding the PTS system glucose-specific EiiCBA component), resulting in 47...
July 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28730473/expression-of-recombinant-phosphoproteins-for-signal-transduction-studies
#6
Karl W Barber, Jesse Rinehart
Complex signaling cascades are difficult to study in vitro without phosphorylated proteins. Here, we describe a technique for the routine production of recombinant phosphoproteins by directly incorporating phosphoserine as a nonstandard amino acid. This protocol utilizes an optimized phosphoserine orthogonal translation system and an engineered strain of E. coli containing no genomic amber codons. This approach has been used to generate a variety of phosphorylated proteins to understand the role of protein phosphorylation in cell signaling...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28721397/hivis-dna-or-hivisopt-dna-priming-followed-by-cmdr-vaccinia-based-boosts-induce-both-humoral-and-cellular-murine-immune-responses-to-hiv
#7
J Hinkula, S Petkov, K Ljungberg, D Hallengärd, A Bråve, M Isaguliants, T Falkeborn, S Sharma, V Liakina, M Robb, M Eller, B Moss, G Biberfeld, E Sandström, C Nilsson, K Markland, P Blomberg, B Wahren
BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated...
June 2017: Heliyon
https://www.readbyqxmd.com/read/28688204/enhancing-expression-of-the-pseudorabies-virus-glycoprotein-e-in-yeast-and-its-application-in-an-indirect-sandwich-elisa
#8
Ching-Ying Wu, Ching-Wei Wu, Chih-Ming Liao, Maw-Sheng Chien, Chienjin Huang
AIMS: The purpose of this study was to produce a recombinant pseudorabies virus (PRV) glycoprotein E (gE) protein with the correct antigenicity for use as a low-cost diagnostic antigen. METHODS AND RESULTS: The gene fragment encoding the amino-terminal immunodominant region of PRV gE (codons 31-270) (gEN31-270) was codon-optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast-expressed gEN31-270 (ygEN31-270) was harvested from the culture supernatant, and ygEN31-270 was shown to exhibit N-linked glycosylation...
July 8, 2017: Journal of Applied Microbiology
https://www.readbyqxmd.com/read/28684180/enhancing-linalool-production-by-engineering-oleaginous-yeast-yarrowia-lipolytica
#9
Xuan Cao, Liu-Jing Wei, Jia-Yu Lin, Qiang Hua
In this study, stepwise increases in linalool production were obtained by combining metabolic engineering and process optimization of an unconventional oleaginous yeast Yarrowia lipolytica. The linalool synthetic pathway was successfully constructed by heterologously expressing a codon-optimized linalool synthase gene from Actinidia arguta in Y. lipolytica. To enhance linalool productivity, key genes involved in the mevalonate pathway were overexpressed in different combinations. Moreover, the overexpression of mutant ERG20(F88W-N119W) gene resulted in further linalool production...
June 23, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28684178/expression-and-characterization-of-the-key-enzymes-involved-in-2-benzoxazolinone-degradation-by-pigmentiphaga-sp-dl-8
#10
Weiliang Dong, Kuan Liu, Jiawei Liu, Zhoukun Shi, Fengxue Xin, Wenming Zhang, Jiangfeng Ma, Hao Wu, Fei Wang, Min Jiang
In this study, the key enzymes involved in 2-benzoxazolinone (BOA) degradation by Pigmentiphaga sp. DL-8 were further verified and characterized in Escherichia coli. By codon optimization and co-expression of molecular chaperones in a combined strategy, recombinant BOA amidohydrolase (rCbaA) and 2-aminophenol (2-AP) 1,2-dioxygenase (rCnbCαCβ) were expressed and purified with the highest activity of 1934.6U·mgprotein(-1) and 32.80U·mgprotein(-1), respectively. BOA could be hydrolyzed to 2AP by rCbaA, which was further transformed to picolinic acid by rCnbCαCβ based on identified catalytic product...
June 23, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28673553/hiv-1-sequence-necessary-and-sufficient-to-package-non-viral-rnas-into-hiv-1-particles
#11
Yang Liu, Olga A Nikolaitchik, Sheikh Abdul Rahman, Jianbo Chen, Vinay K Pathak, Wei-Shau Hu
Genome packaging is an essential step to generate infectious HIV-1 virions and is mediated by interactions between the viral protein Gag and cis-acting elements in the full-length RNA. The sequence necessary and sufficient to allow RNA genome packaging into an HIV-1 particle has not been defined. Here, we used two distinct reporter systems to determine the HIV-1 sequence required for heterologous, non-viral RNAs to be packaged into viral particles. Although the 5' untranslated region (UTR) of the HIV-1 RNA is known to be important for RNA packaging, we found that its ability to mediate packaging relies heavily on the context of the downstream sequences...
June 30, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28671706/an-integrative-machine-learning-strategy-for-improved-prediction-of-essential-genes-in-escherichia-coli-metabolism-using-flux-coupled-features
#12
Sutanu Nandi, Abhishek Subramanian, Ram Rup Sarkar
Prediction of essential genes helps to identify a minimal set of genes that are absolutely required for the appropriate functioning and survival of a cell. The available machine learning techniques for essential gene prediction have inherent problems, like imbalanced provision of training datasets, biased choice of the best model for a given balanced dataset, choice of a complex machine learning algorithm, and data-based automated selection of biologically relevant features for classification. Here, we propose a simple support vector machine-based learning strategy for the prediction of essential genes in Escherichia coli K-12 MG1655 metabolism that integrates a non-conventional combination of an appropriate sample balanced training set, a unique organism-specific genotype, phenotype attributes that characterize essential genes, and optimal parameters of the learning algorithm to generate the best machine learning model (the model with the highest accuracy among all the models trained for different sample training sets)...
July 25, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28664314/avermectin-biosynthesis-stable-functional-expression-of-branched-chain-%C3%AE-keto-acid-dehydrogenase-complex-from-streptomyces-avermitilis-in-escherichia-coli-by-selectively-regulating-individual-subunit-gene-expression
#13
Qianqian Cui, Fengli Zhou, Weifeng Liu, Yong Tao
OBJECTIVE: To improve the production of short branched-chain acyl-CoAs for avermectin biosynthesis, the functional expression of the branched chain α-keto acid dehydrogenase complex (BKDH) from Streptomyces avermitilis was systematically optimized by selectively regulating individual subunit expression in Escherichia coli. RESULTS: Functional expression of the BKDH complex was achieved by independent and selective optimization of individual subunit genes of the complex...
June 29, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28662393/ionic-strength-controlled-hybridization-and-stability-of-hybrids-of-kras-dna-single-nucleotides-a-surface-plasmon-resonance-study
#14
N Giamblanco, S Petralia, S Conoci, C Messineo, G Marletta
The discrimination of a fully matched, unlabeled KRAS wild-type (WT) (C-G) target sample with respect to three of the most frequent KRAS codon mutations (G12 S (C-A), G12 R (C-C), G12C (C-T)) was investigated using an optimized detection strategy involving surface plasmon resonance (SPR), based on optimized probe-surface density and ionic strength control. The changes observed in the SPR signal were always larger for WT compared with the single-mismatch target DNA oligonucleotides, and were aligned with the theoretical energy differences between the base pair C-G, C-T, C-A, C-C...
June 21, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28662217/mistakes-in-translation-reflections-on-mechanism
#15
Yizhou Liu, Joshua S Sharp, Duc H-T Do, Richard A Kahn, Harald Schwalbe, Florian Buhr, James H Prestegard
Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon...
2017: PloS One
https://www.readbyqxmd.com/read/28662040/tuning-the-transcription-and-translation-of-l-amino-acid-deaminase-in-escherichia-coli-improves-%C3%AE-ketoisocaproate-production-from-l-leucine
#16
Yang Song, Jianghua Li, Hyun-Dong Shin, Long Liu, Guocheng Du, Jian Chen
α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from l-leucine, using an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase (l-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of l-AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31...
2017: PloS One
https://www.readbyqxmd.com/read/28660368/high-production-of-genistein-diglucoside-derivative-using-cyclodextrin-glycosyltransferase-from-paenibacillus-macerans
#17
Ruizhi Han, Binbin Ge, Mingyang Jiang, Guochao Xu, Jinjun Dong, Ye Ni
Genistein has been regarded as one important soy isoflavone with multiple health benefits, whereas its applications are limited by the low hydrophilicity. To improve the water solubility, codon optimized cyclodextrin glycosyltransferase from Paenibacillus macerans was employed for genistein transglycosylation in this study. At least four transglycosylation products were produced and identified by HPLC and LC-MS: genistein monoglucoside, diglucoside, triglucoside, and tetraglucoside derivatives. Obviously, the yields of genistein monoglucoside and genistein diglucoside exhibited great superiority compared with other two products...
June 28, 2017: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28658417/evaluation-of-recombinant-cryptosporidium-hominis-gp60-protein-and-anti-gp60-chicken-polyclonal-igy-for-research-and-diagnostic-purposes
#18
Valéria Chamas Miura, Sérgio Moraes Aoki, Paulo Peitl, Lilian Campos Pires, Priscila Dalmagro, Alex Akira Nakamura, Marcelo Vasconcelos Meireles
In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE...
April 2017: Revista Brasileira de Parasitologia Veterinária, Brazilian Journal of Veterinary Parasitology
https://www.readbyqxmd.com/read/28651974/variants-of-ppulcc-a-multi-dye-decolorizing-laccase-from-pleurotus-pulmonarius-expressed-in-pichia-pastoris
#19
Christoph J Behrens, Diana Linke, Aldrige B Allister, Katerina Zelena, Ralf G Berger
A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (-2, -5, -13 AA) of the target protein were combined with different secretion signals...
June 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28650169/increasing-protein-production-rates-can-decrease-the-rate-at-which-functional-protein-is-produced-and-their-steady-state-levels
#20
Ajeet K Sharma, Edward P O'Brien
The rate at which soluble, functional protein is produced by the ribosome has recently been found to vary in complex and unexplained ways as various translation-associated rates are altered through synonymous codon substitutions. To understand this phenomenon, here, we combine a well-established ribosome-traffic model with a master-equation model of cotranslational domain folding to explore the scenarios that are possible for the protein production rate, J, and the functional-nascent protein production rate, F, as the rates of various translation processes are altered for five different E...
July 7, 2017: Journal of Physical Chemistry. B
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