An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

Ma, Pikyee; Varela, Filipa; Magoch, Małgorzata; Silva, Ana Rita; Rosario, Ana Lucia; Brito, José A.; Oliveira, Tânia F.; Nogly, Przemyslaw; Pessanha, Miguel; Stelter, Meike; Kletzin, Arnulf; Henderson, Peter J. F.; Archer, Margarida

doi:10.1371/journal.pone.0076913

Cited by 22 publications

(18 citation statements)

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“…Cloning and amplification of expression of the PucI protein in E. coli was achieved using a strategy that we and others have found successful with a wide range of bacterial and archaeal membrane proteins (Ward et al, 1999;Saidijam et al, 2003;Szakonyi et al, 2007;Ma et al, 2008Ma et al, , 2013Bettaney et al, 2013). This involved design of PCR primers (forward, 59-CCGGAATTCGCATATGAAA-TTAAAAGAGAGTCAGCAGCAATCCA-39, and reverse, 59-AAAAC-TGCAGCTTCAGCCTGGCGGACCTGCGCATGTT-39) to extract and amplify the pucI gene from B. subtilis genomic DNA, introducing EcoRI and Pst I restriction sites at the 59 and 39 ends, respectively.…”

Section: Methodsmentioning

confidence: 99%

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

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Иванова

et al. 2016

Microbiology

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This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning a-helices with both N-and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His 6 ) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of 14 C-allantoin into energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (K m app) of 24¡3 mM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His 6 ) was solubilized from inner membranes using n-dodecyl-b-D-maltoside and purified. The isolated protein contained a substantial proportion of a-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

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Section: Methodsmentioning

confidence: 99%

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

Patching

Иванова

et al. 2016

Microbiology

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“…a family of proteins or the proteome from a single organism), which are reminiscent of laboratory-scale experiments that precede structural or biochemical analyses. While a number of datasets exist (5, 46-57), we identified seven for which complete sequence information could be obtained to calculate all the necessary sequence features (46)(47)(48)(49)(50)(51)(52).…”

Section: Further Demonstration Of Prediction Against Small-scale Indementioning

confidence: 99%

“…2b), of the various data sets, only the expression of archaeal transporters presents quantitative outcomes for consideration. For this dataset, 14 archaeal transporters were chosen based on their homology to human proteins (46) and tested for expression in E. coli; total protein was quantified in the membrane fraction by Coomassie Blue staining of an SDS-PAGE gel. Here, the majority of the expressing proteins fall into the higher half of the IMProve scores, 7 out of 9 of those with multiple positive outcomes (Fig.…”

Section: Further Demonstration Of Prediction Against Small-scale Indementioning

confidence: 99%

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A statistical model for improved membrane protein expression using sequence-derived features

Saladi

Javed

Müller

et al. 2018

Journal of Biological Chemistry

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The heterologous expression of integral membrane proteins (IMPs) remains a major bottleneck in the characterization of this important protein class. IMP expression levels are currently unpredictable, which renders the pursuit of IMPs for structural and biophysical characterization challenging and inefficient. Experimental evidence demonstrates that changes within the nucleotide or amino-acid sequence for a given IMP can dramatically affect expression levels; yet these observations have not resulted in generalizable approaches to improve expression levels. Here, we develop a data-driven statistical predictor named IMProve, that, using only sequence information, increases the likelihood of selecting an IMP that expresses in E. coli. The IMProve model, trained on experimental data, combines a set of sequencederived features resulting in an IMProve score, where higher values have a higher probability of success. The model is rigorously validated against a variety of independent datasets that contain a wide range of experimental outcomes from various IMP expression trials. The results demonstrate that use of the model can more than double the number of successfully expressed targets at any experimental scale. IMProve can immediately be used to identify favorable targets for characterization. Most notably, IMProve demonstrates for the first time that IMP expression levels can be predicted directly from sequence.

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“…85,86 Nas figuras abaixo podemos visualizar e comparar a expressão da SelT nas cepas BL21 (DE3) e C43 (DE3) ambas de E.…”

Section: A) Alteração Da Cepa De Expressãounclassified

Selenoproteínas: Seril-tRNA Sintetase e as selenoproteínas do Trypanosoma brucei

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AGRADECIMENTOSUma tese de doutoramento é uma longa viagem, com muitos percalços pelo caminho. E para a realização deste trabalho obtive inúmeros contributos de naturezas diversas que não podem e nem devem deixar de serem realçados. Por essa razão, desejo expressar os meus sinceros agradecimentos:Ao meu orientador Prof. Dr. Otavio H. Thiemann, pela competência científica e acompanhamento do trabalho, pela disponibilidade e generosidade, pelas críticas, correções e sugestões relevantes feitas durante a orientação e acima de tudo pela grande amizade cultivada durante estes anos de parceria. Aos amigos do Laboratório de Biologia Estruturalque sempre me ajudaram diretamente ou indiretamente. Em especial aos alunos do "U do Otavio" e em particular à Nath por deixar eu "tentar orientá-la" durante sua iniciação científica. E às amigas especiais: Cy, Ju Torini, Livia Manzine e Simone.À toda a equipe da biblioteca do IFSC, pelo trabalho na verificação da formatação do texto para que ele estivesse dentro das normas acadêmicas vigentes, também expresso meus agradecimentos. À todos os funcionários do IFSC, de maneira especial aos da biblioteca, serviço de pós-graduação e da área financeira que sempre foram acessíveis quando procurei.Aos meus pais, meus maiores mestres, Luiz e Maria do Carmo que dignamente me apresentaram à importância da família e o caminho da honestidade e persistência.Ao Samuel, meu marido, e Mariah, minha filha, que são meus grandes companheiros, amigos e meus amores pelo apoio incondicional em todos os momentos, principalmente nos de incerteza, muito comuns para quem tenta trilhar novos caminhos. Sem vocês nenhuma conquista valeria à pena.À uma força maior que sempre me acompanha: Deus.Ao CNPq pelo apoio financeiro.

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An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

Cited by 22 publications

References 44 publications

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

A statistical model for improved membrane protein expression using sequence-derived features

Selenoproteínas: Seril-tRNA Sintetase e as selenoproteínas do Trypanosoma brucei

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