Assuntos
Cegueira/história , Canadá , História do Século XVIII , História do Século XIX , Humanos , Filatelia , Remissão EspontâneaRESUMO
A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.
Assuntos
Genes Sintéticos , Receptores da Eritropoetina/biossíntese , Sequência de Bases , Western Blotting , Clonagem Molecular/métodos , Códon , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Genes Bacterianos , Vetores Genéticos , Glicosilação , Humanos , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Sinais Direcionadores de Proteínas , Receptores da Eritropoetina/isolamento & purificação , Receptores da Eritropoetina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Streptomyces/genéticaRESUMO
An extracellular tripeptidyl aminopeptidase has been purified from Streptomyces lividans 66 cell-free cultures. The enzyme is a major component of the secreted proteolytic activity. The protease removes only the N-terminal tripeptide from recombinant human GM-CSF and IL-3 but does not cleave recombinant human IL-6. The enzyme cleaves the synthetic tripeptide substrates APA-pNA and APM-pNA but does not cleave substrates with blocked amino terminals. Smaller substrates are not cleaved. The enzyme appears to be a serine protease of 55 kDa molecular weight. The pH optimum is between 7.5 and 8.5 but varies slightly with the substrate. The N-terminal sequence and amino acid composition have been determined.
