RESUMO
Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.
Assuntos
Fenilalanina-tRNA Ligase/genética , Fenilalanina-tRNA Ligase/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Haemophilus influenzae/enzimologia , Cinética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Óperon/genética , Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Staphylococcus aureus/genética , Streptococcus pneumoniae/enzimologia , Relação Estrutura-Atividade , Thermus thermophilus/enzimologiaRESUMO
Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.
Assuntos
Proteínas de Choque Térmico , Proteínas Periplásmicas , Serina Endopeptidases/química , Serina Endopeptidases/genética , Alanina/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caseínas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Fibroblastos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Temperatura Alta , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Proteínas Mitocondriais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Presenilina-1 , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina/química , Serina Endopeptidases/biossíntese , Frações Subcelulares/metabolismo , Temperatura , Fatores de Tempo , Distribuição Tecidual , Tunicamicina/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.
Assuntos
Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Baculoviridae/genética , Caseínas/química , Escherichia coli/enzimologia , Escherichia coli/genética , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Proteínas Mitocondriais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.