Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.Universidade Federal de São Paulo (UNIFESP) Departamento de Ciências BiológicasCentro Latino-Americano e do Caribe de Informações em Ciências da SaúdeUNIFESP, Depto. de Ciências BiológicasSciEL

Avaliação da produção científica e o Projeto SciELO*

Partindo da análise da produção científica brasileira e da carência de bases de dados em informação científica, o autor aborda o Projeto SciELO - Scientific Electronic Library Online - como instrumento para tornar a produção nacional mais visível e acessível via meio eletrônico e, ao mesmo tempo, criar uma base de dados pela qual seja possível avaliar a produção científica do país e aumentar a sua visibilidade internacional

The structural molecular biology network of the State of São Paulo, Brazil

This article describes the achievements of the Structural Molecular Biology Network (SMolBNet), a collaborative program of structural molecular biology, centered in the State of São Paulo, Brazil, and supported by São Paulo State Funding Agency (FAPESP). It gathers twenty scientific groups and is coordinated by the scientific staff of the Center of Structural Molecular Biology, at the National Laboratory of Synchrotron Light (LNLS), in Campinas. The SMolBNet program has been aimed at 1) solving the structure of proteins of interest related to the research projects of the groups. In some cases, the choice has been to select proteins of unknown function or of possible novel structure obtained from the sequenced genomes of the FAPESP genomic program; 2) providing the groups with training in all the steps of the protein structure determination: gene cloning, protein expression, protein purification, protein crystallization and structure determination. Having begun in 2001, the program has been successful in both aims. Here, four groups reveal their participation in the program and describe the structural aspects of the proteins they have selected to study.Esse artigo descreve realizações do Programa SMolBNet (Rede de Biologia Molecular Estrutural) do Estado de São Paulo, apoiado pela FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo). Ele reúne vinte grupos de pesquisa e é coordenado pelos pesquisadores do Laboratório Nacional de Luz Síncrotron (LNLS), em Campinas. O Programa SMolBNet tem como metas: Elucidar a estrutura tridimensional de proteínas de interesse aos grupos de pesquisa componentes do Programa; Prover os grupos com treinamento em todas as etapas de determinação de estrutura: clonagem gênica, expressão de proteínas, purificação de proteínas, cristalização de proteínas e elucidação de suas estruturas. Tendo começado em 2001, o Programa alcançou sucesso em ambas as metas. Neste artigo, quatro dos grupos descrevem suas participações, e discutem aspectos estruturais das proteínas que eles selecionaram para estudos.Centro de Biologia Molecular Estrutural Laboratório Nacional de Luz SíncrotronUniversidade de São Paulo Instituto de Biociências Departamento de Genética e Processos EvolutivosUniversidade de São Paulo Instituto de Química Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaCentro Latino-Americano e do Caribe de Informação em Ciências da Saúde BIREMEUNIFESP, EPMSciEL

Proteome of the phytopathogen Xanthomonas citri subsp. citri: a global expression profile

<p>Abstract</p> <p>Background</p> <p>Citrus canker is a disease caused by <it>Xantomonas citri </it>subsp.<it>citri (Xac)</it>, and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of <it>Xac </it>was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).</p> <p>Results</p> <p>In order to gain insight into the metabolism of <it>Xac</it>, cells were grown on two different media (<b>NB </b>- Nutrient Broth and <b>TSE </b>- Tryptone Sucrose broth enriched with glutamic acid), and proteins were proteolyzed with trypsin and examined by 2D LC-MS/MS. Approximately 39% of all predicted proteins by annotation of <it>Xac </it>were identified with their component peptides unambiguously assigned to tandem mass spectra. The proteins, about 1,100, were distributed in all annotated functional categories.</p> <p>Conclusions</p> <p>This is the first proteomic reference map for the most aggressive strain of <it>Xanthomonas </it>pathogen of all orange varieties. The compilation of metabolic pathways involved with bacterial growth showed that <it>Xac </it>expresses a complete central and intermediary metabolism, replication, transcription and translation machineries and regulation factors, distinct membrane transporters (ABC, MFS and pumps) and receptors (MCP, TonB dependent and metabolites acquisition), two-component systems (sensor and regulatory components) and response regulators. These data corroborate the growth curve <it>in vitro </it>and are the first reports indicating that many of these genome annotated genes are translated into operative in <it>Xac</it>. This proteomic analysis also provided information regarding the influence of culture medium on growth and protein expression of <it>Xac</it>.</p

La evaluación de la producción científica y el Proyecto SciELO

Beginning with the anlysis of the state of the Brazilian scientific literature and the lack of bibliographical databases in the field of scientific information, this lecture examines the SciELO Project (Scientific Electronic Library Online) as a tool to make the scientific literature more accesible by way of electronic means, as well a create a database that enables the evaluation of this literature in the country, and the increase of its international visibility