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[PMID]:28911099
[Au] Autor:Le S; Serrano E; Kawamura R; Carrasco B; Yan J; Alonso JC
[Ad] Endereço:Department of Physics, National University of Singapore, 117551, Singapore.
[Ti] Título:Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.
[So] Source:Nucleic Acids Res;45(15):8873-8885, 2017 Sep 06.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.
[Mh] Termos MeSH primário: Bacillus subtilis/genética
Proteínas de Bactérias/genética
Proteínas de Ligação a DNA/genética
Regulação Bacteriana da Expressão Gênica
Proteínas de Membrana/genética
Recombinases Rec A/genética
Transformação Bacteriana
[Mh] Termos MeSH secundário: Trifosfato de Adenosina/metabolismo
Bacillus subtilis/metabolismo
Proteínas de Bactérias/metabolismo
Cromossomos Bacterianos/química
Cromossomos Bacterianos/metabolismo
DNA Bacteriano/genética
DNA Bacteriano/metabolismo
DNA de Cadeia Simples/genética
DNA de Cadeia Simples/metabolismo
Proteínas de Ligação a DNA/metabolismo
Cinética
Proteínas de Membrana/metabolismo
Plasmídeos/química
Plasmídeos/metabolismo
Recombinases Rec A/metabolismo
Recombinação Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA, Bacterial); 0 (DNA, Single-Stranded); 0 (DNA-Binding Proteins); 0 (DprA protein, bacteria); 0 (Membrane Proteins); 0 (RecX protein, Xanthomonas campestris); 8L70Q75FXE (Adenosine Triphosphate); EC 2.7.7.- (Rec A Recombinases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171012
[Lr] Data última revisão:
171012
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170916
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx583


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[PMID]:28902855
[Au] Autor:Claassens NJ; Siliakus MF; Spaans SK; Creutzburg SCA; Nijsse B; Schaap PJ; Quax TEF; van der Oost J
[Ad] Endereço:Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.
[Ti] Título:Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms.
[So] Source:PLoS One;12(9):e0184355, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool.
[Mh] Termos MeSH primário: Algoritmos
Códon/genética
Escherichia coli
Regulação Bacteriana da Expressão Gênica
Proteínas de Membrana/biossíntese
Software
[Mh] Termos MeSH secundário: Escherichia coli/genética
Escherichia coli/metabolismo
Código Genético
Proteínas de Membrana/genética
Engenharia Metabólica/métodos
Organismos Geneticamente Modificados
Proteínas Recombinantes/biossíntese
Proteínas Recombinantes/genética
Transcrição Genética/genética
Transformação Bacteriana/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Codon); 0 (Membrane Proteins); 0 (Recombinant Proteins)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170914
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0184355


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[PMID]:28857325
[Au] Autor:Magaña Vergara C; Kallenberg CJL; Rogasch M; Hübner CG; Song YH
[Ad] Endereço:Institute of Physics, University of Luebeck, Ratzeburger Allee 160, Luebeck, 23562, Germany.
[Ti] Título:A versatile vector for mycobacterial protein production with a functional minimized acetamidase regulon.
[So] Source:Protein Sci;26(11):2302-2311, 2017 Nov.
[Is] ISSN:1469-896X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Recombinant protein expression is a prerequisite for diverse investigations of proteins at the molecular level. For targets from Mycobacterium tuberculosis it is favorable to use M. smegmatis as an expression host, a species from the same genus. In the respective shuttle vectors, target gene expression is controlled by the complex tetra-cistronic acetamidase regulon. As a result, the size of those vectors is large, rendering them of limited use, especially when the target proteins are expressed from multi-cistronic operons. Therefore, in the current work we present a versatile new expression vector in which the acetamidase regulon has been minimized by deleting the two genes amiD and amiS. We assessed the functional properties of the resulting vector pMyCA and compared it with those of the existing vector pMyNT that contains the full-length acetamidase regulon. We analyzed the growth features and protein expression patterns of M. smegmatis cultures transformed with both vectors. In addition, we created mCherry expression constructs to spectroscopically monitor the expression properties of both vectors. Our experiments showed that the minimized vector exhibited several advantages over the pMyNT vector. First, the overall yield of expressed protein is higher due to the higher yield of bacterial mass. Second, the heterologous expression was regulated more tightly, offering an expression tool for diverse target proteins. Third, it is suitable for large multi-protein complexes that are expressed from multi-cistronic operons. Additionally, our results propose a new understanding of the regulation mechanism of the acetamidase regulon with the potential to construct more optimized vectors in the future.
[Mh] Termos MeSH primário: Amidoidrolases/genética
Proteínas de Bactérias/genética
Regulação Bacteriana da Expressão Gênica
Vetores Genéticos/química
Mycobacterium smegmatis/genética
Mycobacterium tuberculosis/genética
Regulon
[Mh] Termos MeSH secundário: Amidoidrolases/metabolismo
Proteínas de Bactérias/metabolismo
Sequência de Bases
Deleção de Genes
Genes Reporter
Vetores Genéticos/metabolismo
Proteínas Luminescentes/genética
Proteínas Luminescentes/metabolismo
Mycobacterium smegmatis/metabolismo
Mycobacterium tuberculosis/metabolismo
Óperon
Regiões Promotoras Genéticas
Proteínas Recombinantes/biossíntese
Proteínas Recombinantes/genética
Proteínas Recombinantes/isolamento & purificação
Transformação Bacteriana
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Luminescent Proteins); 0 (Recombinant Proteins); 0 (red fluorescent protein); EC 3.5.- (Amidohydrolases); EC 3.5.1.- (acetamidase)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171103
[Lr] Data última revisão:
171103
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170901
[St] Status:MEDLINE
[do] DOI:10.1002/pro.3288


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[PMID]:28771515
[Au] Autor:Leong CG; Bloomfield RA; Boyd CA; Dornbusch AJ; Lieber L; Liu F; Owen A; Slay E; Lang KM; Lostroh CP
[Ad] Endereço:Department of Molecular Biology, Colorado College, Colorado Springs, Colorado, United States of America.
[Ti] Título:The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi.
[So] Source:PLoS One;12(8):e0182139, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Here we present an examination of type IV pilus genes associated with competence and twitching in the bacterium Acinetobacter baylyi (strain ADP1, BD413). We used bioinformatics to identify potential competence and twitching genes and their operons. We measured the competence and twitching phenotypes of the bioinformatically-identified genes. These results demonstrate that competence and twitching in A. baylyi both rely upon a core of the same type IV pilus proteins. The core includes the inner membrane assembly platform (PilC), a periplasmic assemblage connecting the inner membrane assembly platform to the secretin (ComM), a secretin (ComQ) and its associated pilotin (PilF) that assists with secretin assembly and localization, both cytoplasmic pilus retraction ATPases (PilU, PilT), and pilins (ComP, ComB, PilX). Proteins not needed for both competence and twitching are instead found to specialize in either of the two traits. The pilins are varied in their specialization with some required for either competence (FimT) and others for twitching (ComE). The protein that transports DNA across the inner membrane (ComA) specializes in competence, while signal transduction proteins (PilG, PilS, and PilR) specialize in twitching. Taken together our results suggest that the function of accessory proteins should not be based on homology alone. In addition the results suggest that in A. baylyi the mechanisms of natural transformation and twitching are mediated by the same set of core Type IV pilus proteins with distinct specialized proteins required for each phenotype. Finally, since competence requires multiple pilins as well as both pilus retraction motors PilU and PilT, this suggests that A. baylyi employs a pilus in natural transformation.
[Mh] Termos MeSH primário: Acinetobacter/genética
Acinetobacter/metabolismo
Proteínas de Fímbrias/metabolismo
Transformação Bacteriana/genética
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Proteínas de Bactérias/metabolismo
DNA Bacteriano/isolamento & purificação
DNA Bacteriano/metabolismo
Proteínas de Fímbrias/química
Proteínas de Fímbrias/genética
Fímbrias Bacterianas/genética
Teste de Complementação Genética
Proteínas de Membrana Transportadoras/química
Proteínas de Membrana Transportadoras/genética
Proteínas de Membrana Transportadoras/metabolismo
Fenótipo
Alinhamento de Sequência
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA, Bacterial); 0 (Membrane Transport Proteins); 147680-16-8 (Fimbriae Proteins)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171013
[Lr] Data última revisão:
171013
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170804
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0182139


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[PMID]:28693663
[Au] Autor:Li T; Xu M; Zheng L
[Ad] Endereço:Department of Microbiology and Parasitology, College of Basic Medical Science, China Medical University, No. 77 Puhe Road, Shenyang 110122, Liaoning Province, PR China.
[Ti] Título:Is SpxA2 involved in hydrogen peroxide production and competence development in Streptococcus sanguinis?
[So] Source:J Med Microbiol;66(7):981-989, 2017 Jul.
[Is] ISSN:1473-5644
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:PURPOSE: The objective of the present study was to investigate whether Streptococcus sanguinis SpxA2 plays a role in competence development and endogenous H2O2 generation, and whether the SpxA2 Cys10-XX-Cys13 (CXXC) motif is involved in competence development. METHODOLOGY: The competence development of wild-type S. sanguinis (SK36) and its derivatives was compared by transformation efficiency assay and real-time RT-PCR. The spx allele mutants, spxA2 (C10A) and spxA2 (C13A), were constructed by site-directed mutagenesis. The Δpox mutant was treated with 1 mM H2O2 to exclude the effect of other Pox products on competence development. RESULTS: Compared with the wild-type (4.42±0.58×10-4), the ΔspxA2 mutant showed decreased transformation efficiency (0.07±0.03×10-4). Furthermore, there was a 2- to 15-fold reduction in ΔspxA2 mutant com gene expression. SpxA2 was able to down-regulate endogenous H2O2 generation by repressing pox expression. Additionally, endogenous H2O2 negatively regulated competence without affecting spxA2 expression. The Δpox mutant increased com gene expression (2- to 8-fold), but the 1 mM H2O2-treated Δpox mutant showed decreased com gene expression. Interestingly, the ΔspxA2Δpox mutant showed enhanced competence-associated parameters. The fact that spxA2 (C10A) and spxA2 (C13A) behaved like the ΔspxA2 mutant revealed the role of the CXXC motif in competence development. CONCLUSION: Although the intricate relationship between SpxA2, pox-mediated H2O2 production and competence development was clarified in S. sanguinis, it would be worthwhile to explore further whether H2O2 is involved in competence development through oxidizing the SpxA2 CXXC motif.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Competência de Transformação por DNA
Peróxido de Hidrogênio/metabolismo
Streptococcus sanguis/genética
Streptococcus sanguis/metabolismo
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Análise Mutacional de DNA
Deleção de Genes
Mutagênese Sítio-Dirigida
Oxidantes/metabolismo
Reação em Cadeia da Polimerase em Tempo Real
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Transformação Bacteriana
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Oxidants); BBX060AN9V (Hydrogen Peroxide)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170809
[Lr] Data última revisão:
170809
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170712
[St] Status:MEDLINE
[do] DOI:10.1099/jmm.0.000506


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[PMID]:28690319
[Au] Autor:Veening JW; Blokesch M
[Ad] Endereço:Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Present address: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, CH-1015 Lausanne, Switzerland.
[Ti] Título:Interbacterial predation as a strategy for DNA acquisition in naturally competent bacteria.
[So] Source:Nat Rev Microbiol;15(10):621-629, 2017 10.
[Is] ISSN:1740-1534
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Natural competence enables bacteria to take up exogenous DNA. The evolutionary function of natural competence remains controversial, as imported DNA can act as a source of substrates or can be integrated into the genome. Exogenous homologous DNA can also be used for genome repair. In this Opinion article, we propose that predation of non-related neighbouring bacteria coupled with competence regulation might function as an active strategy for DNA acquisition. Competence-dependent kin-discriminated killing has been observed in the unrelated bacteria Vibrio cholerae and Streptococcus pneumoniae. Importantly, both the regulatory networks and the mode of action of neighbour predation differ between these organisms, with V. cholerae using a type VI secretion system and S. pneumoniae secreting bacteriocins. We argue that the forced release of DNA from killed bacteria and the transfer of non-clonal genetic material have important roles in bacterial evolution.
[Mh] Termos MeSH primário: Competência de Transformação por DNA/genética
DNA Bacteriano/genética
Transferência Genética Horizontal/genética
Streptococcus pneumoniae/genética
Transformação Bacteriana/genética
Vibrio cholerae/genética
[Mh] Termos MeSH secundário: Transporte Biológico
Regulação Bacteriana da Expressão Gênica
Sistemas de Secreção Tipo VI/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Type VI Secretion Systems)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:171121
[Lr] Data última revisão:
171121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170711
[St] Status:MEDLINE
[do] DOI:10.1038/nrmicro.2017.66


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[PMID]:28660315
[Au] Autor:Vitali J; Singh AK; Colaneri MJ
[Ad] Endereço:Department of Physics, Cleveland State University, Cleveland, OH, 44115, USA. j.vitali@csuohio.edu.
[Ti] Título:Characterization of the Dihydroorotase from Methanococcus jannaschii.
[So] Source:Protein J;36(4):361-373, 2017 Aug.
[Is] ISSN:1875-8355
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC-MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with V = 12.2 µmol/min/mg and K = 0.14 mM at 25 °C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85 and 90 °C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85 °C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability-more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined.
[Mh] Termos MeSH primário: Proteínas Arqueais/química
Di-Hidro-Orotase/química
Methanocaldococcus/química
Zinco/química
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Proteínas Arqueais/genética
Proteínas Arqueais/metabolismo
Bacillus anthracis/química
Bacillus anthracis/enzimologia
Sítios de Ligação
Clonagem Molecular
Sequência Conservada
Di-Hidro-Orotase/genética
Di-Hidro-Orotase/metabolismo
Estabilidade Enzimática
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Cinética
Methanocaldococcus/enzimologia
Peso Molecular
Fases de Leitura Aberta
Plasmídeos/química
Plasmídeos/metabolismo
Ligação Proteica
Conformação Proteica em alfa-Hélice
Conformação Proteica em Folha beta
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Alinhamento de Sequência
Relação Estrutura-Atividade
Especificidade por Substrato
Termodinâmica
Transformação Bacteriana
Zinco/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Archaeal Proteins); 0 (Recombinant Proteins); EC 3.5.2.3 (Dihydroorotase); J41CSQ7QDS (Zinc)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171004
[Lr] Data última revisão:
171004
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170630
[St] Status:MEDLINE
[do] DOI:10.1007/s10930-017-9729-7


  8 / 5795 MEDLINE  
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[PMID]:28639160
[Au] Autor:Guan L; Chen L; Chen Y; Zhang N; Han Y
[Ad] Endereço:College of Food and Bioengineering, Zhengzhou University of Light Industry, Number 5, Dongfeng Road, Zhengzhou, 450002, China.
[Ti] Título:Expression and Activity Analysis of Fructosyltransferase from Aspergillus oryzae.
[So] Source:Protein J;36(4):352-360, 2017 Aug.
[Is] ISSN:1875-8355
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368 bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-ß-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45 kDa. The optimal conditions of protein expression were 25 °C, 0.1 mM IPTG, and 8 h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5 M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.
[Mh] Termos MeSH primário: Aspergillus oryzae/química
Proteínas Fúngicas/genética
Hexosiltransferases/genética
Plasmídeos/metabolismo
Sacarose/análogos & derivados
[Mh] Termos MeSH secundário: Aspergillus oryzae/enzimologia
Sequência de Bases
Clonagem Molecular
Escherichia coli/genética
Escherichia coli/metabolismo
Proteínas Fúngicas/metabolismo
Expressão Gênica
Hexosiltransferases/metabolismo
Corpos de Inclusão/química
Corpos de Inclusão/efeitos dos fármacos
Peso Molecular
Fases de Leitura Aberta
Plasmídeos/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Alinhamento de Sequência
Sacarose/metabolismo
Transformação Bacteriana
Ureia/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fungal Proteins); 0 (Recombinant Proteins); 0 (sucrose-6-acetate); 57-50-1 (Sucrose); 8W8T17847W (Urea); EC 2.4.1.- (Hexosyltransferases); EC 2.4.1.9 (inulosucrase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170728
[Lr] Data última revisão:
170728
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170623
[St] Status:MEDLINE
[do] DOI:10.1007/s10930-017-9725-y


  9 / 5795 MEDLINE  
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[PMID]:28589291
[Au] Autor:Shi J; Ma X; Gao Y; Fan D; Zhu C; Mi Y; Xue W
[Ad] Endereço:Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Taibai North Road 229, Xi'an, 710069, Shaanxi, China.
[Ti] Título:Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with a Viral Prolyl 4-Hydroxylase in Escherichia coli.
[So] Source:Protein J;36(4):322-331, 2017 Aug.
[Is] ISSN:1875-8355
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:High-level expression of recombinant collagen by genetic engineering is urgently required. Recombinant collagen is different from natural collagen in its hydroxyproline (Hyp) content and thermal stability. To obtain hydroxylated collagen for applications in biomedicine and biomaterials, the human collagen α1(III) chain was co-expressed with the viral prolyl 4-hydroxylase A085R in Escherichia coli. Unlike previous reports using human prolyl 4-hydroxylase, this study examined the hydroxylation of full-length human collagen α1(III) chain (COL3A1) by viral prolyl 4-hydroxylase. The genes encoding these two proteins were controlled by different promoters, Ptac and PRPL, on a recombinant pKK223-3 plasmid. The sequencing results verified that the target genes were successfully inserted into the recombinant vector. Based on quantitative PCR, SDS-PAGE, and western blotting, successful expression by E. coli BL21(DE3) was detected at the mRNA and protein levels for both loci. Liquid chromatography-mass spectrometry (LC-MS/MS) results suggested that the highest Hyp yield was obtained when the two proteins were induced with 0.5 mM IPTG and heat-shock treatment at 50 °C, corresponding to high enzyme expression and low human collagen α1(III) chain expression levels. A biological activity analysis indicated that the recombinant collagen with the highest hydroxylation level supported the growth of baby hamster kidney cells, similar to observations for native collagen. The production of hydroxylated collagen in this study establishes a new method for collagen hydroxylation and provides a basis for the application of recombinant collagen expressed in E. coli.
[Mh] Termos MeSH primário: Colágeno Tipo III/metabolismo
Escherichia coli/metabolismo
Plasmídeos/metabolismo
Prolil Hidroxilases/metabolismo
Proteínas Recombinantes/metabolismo
Proteínas Virais/metabolismo
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Animais
Linhagem Celular
Proliferação Celular/efeitos dos fármacos
Colágeno Tipo III/química
Colágeno Tipo III/genética
Colágeno Tipo III/farmacologia
Cricetinae
Escherichia coli/genética
Expressão Gênica
Seres Humanos
Hidroxilação
Phycodnaviridae/química
Phycodnaviridae/enzimologia
Plasmídeos/química
Prolil Hidroxilases/genética
Regiões Promotoras Genéticas
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/farmacologia
Alinhamento de Sequência
Transformação Bacteriana
Proteínas Virais/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (COL3A1 protein, human); 0 (Collagen Type III); 0 (Recombinant Proteins); 0 (Viral Proteins); EC 1.14.11.- (Prolyl Hydroxylases)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170728
[Lr] Data última revisão:
170728
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170608
[St] Status:MEDLINE
[do] DOI:10.1007/s10930-017-9723-0


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[PMID]:28575400
[Au] Autor:Dalia TN; Yoon SH; Galli E; Barre FX; Waters CM; Dalia AB
[Ad] Endereço:Department of Biology, Indiana University, Bloomington, IN 47401, USA.
[Ti] Título:Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases.
[So] Source:Nucleic Acids Res;45(12):7527-7537, 2017 Jul 07.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
[Mh] Termos MeSH primário: Proteínas de Bactérias/genética
Exonucleases/genética
Edição de Genes/métodos
Genoma Bacteriano
Transformação Bacteriana
[Mh] Termos MeSH secundário: Acinetobacter/genética
Acinetobacter/metabolismo
Proteínas de Bactérias/antagonistas & inibidores
Proteínas de Bactérias/metabolismo
Reparo de Erro de Pareamento de DNA
DNA Bacteriano/genética
DNA Bacteriano/metabolismo
DNA de Cadeia Simples/genética
DNA de Cadeia Simples/metabolismo
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Exonucleases/antagonistas & inibidores
Exonucleases/deficiência
Recombinação Homóloga
Reação em Cadeia da Polimerase Multiplex/métodos
Mutação
Fósforo-Oxigênio Liases/genética
Fósforo-Oxigênio Liases/metabolismo
Vibrio cholerae/genética
Vibrio cholerae/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA, Bacterial); 0 (DNA, Single-Stranded); 0 (Escherichia coli Proteins); EC 3.1.- (Exonucleases); EC 4.6.- (Phosphorus-Oxygen Lyases); EC 4.6.1.- (diguanylate cyclase)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171017
[Lr] Data última revisão:
171017
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170603
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx496



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