RESUMO
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.
Assuntos
Humanos , Chaperonina 10 , Chaperonina 60 , Escherichia coli/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Chaperonina 10 , Chaperonina 60 , Cromatografia por Troca Iônica , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Expressão Gênica , Proteína 1 Reguladora do Ferro/isolamento & purificação , Proteínas de Ligação a RNA , SolubilidadeRESUMO
BACKGROUND & OBJECTIVE: We report a new polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) assay using mycobacterial groES as a target to identify Mycobacterium avium and M. intracellulare in clinical samples. METHODS: The assay was standardized using M. avium and M. intracellulare standard strains obtained from ATCC and was tested with 45 M. avium-M. intracellulare complex (MAC) clinical isolates (Of which 31 were from HIV(+) individuals). The standard and clinical strains were typed with HPLC based mycolic acid fingerprinting. RESULTS: Three polymorphisms (BamHI, BstNI and HgaI) were identified for inter-species differentiation among standard strains; of which, only HgaI was found to be useful in clinical isolates. Of the 45 isolates, 25 were M. avium and 20 were M. intracelluare. MAC isolates, which could not be differentiated by HPLC analysis, were also typed by this method. INTERPRETATION & CONCLUSION: The use of mycobacterial groES as a PCR-RFLP target for M. avium and M. intracellulare is a simple and rapid method that can complement HPLC in their differentiation.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Chaperonina 10/genética , Proteínas de Choque Térmico/genética , Humanos , Mycobacterium avium/genética , Complexo Mycobacterium avium/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
<p><b>OBJECTIVE</b>To obtain Chlamydia trachomatis heat shock protein (cHSP) 10 gene from clinical secretion samples.</p><p><b>METHODS</b>cHSP10 gene was amplified from 20 cases of clinical secretion samples with positive gold-labelling by specific primers of cHSP10 and identified by sequence analysis.</p><p><b>RESULTS</b>cHSP10 full-length gene was amplified from 1 of 20 cases of clinical secretion samples with positive gold-labelling. cHSP10 gene encoding 102 amino acids contains 306 bp, which nuclotide at position 194 changes from T to A, leading to the change of corresponding amino acid.</p><p><b>CONCLUSIONS</b>cHSP10 gene may be cloned from clinical secretion samples with positive gold-labelling, which make it possible to further construct expression plasmid of recombinant cHSP10.</p>
Assuntos
Feminino , Humanos , Masculino , Anticorpos Antibacterianos , Sangue , Proteínas de Bactérias , Genética , Sequência de Bases , Chaperonina 10 , Química , Genética , Alergia e Imunologia , Chlamydia trachomatis , Química , Alergia e Imunologia , Clonagem Molecular , Infertilidade Feminina , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA imcompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submitidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüênciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 710 resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado...
Assuntos
Blastocladiella , Chaperonina 10 , Chaperonina 60 , Técnicas In Vitro , Proteínas de Choque Térmico HSP90/biossíntese , RNA Mensageiro , Vetores Genéticos/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Northern Blotting , Western Blotting , Reação em Cadeia da Polimerase/métodosRESUMO
The response to recombinant 10-KD heat shock protein (HSP of Mycobacterium leprae (rML10) was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls a leprosy-endemic area in tne North East of Argentina. Some technical parameters were a analyzed: within-assay and between-assay variability, dose-response curves and dectability indexed (specificity and sensitivity) of ELISA applied to measure anti-10kDa antibodies. High levels of these antibodies have already been reported in positive baciloscopy patients; herein we have also demonstrated that tuberculosis patients sera cross-react with this M. leprae antigen. This test seems to have a low sensificity for leporsy detection; it confirms that antibodies against highly conserved HSP antigens are important in the polycional response against mycobacterial epitopes in leprosy as well as in tuberculosis.