RESUMO
Urate oxidase, an enzyme used in human therapy, is currently produced industrially by a strain of Aspergillus flavus. Two strategies of strain improvement were tested in order to obtain higher yields of urate oxidase. The first one, based on a classical mutation-selection protocol, led to the isolation of a mutant strain that overproduced uricase two-fold as compared to the industrial strain. The second one consisted in the construction of transformed strains that had integrated multiple copies of a urate oxidase-expression vector. A twenty-fold improvement in urate oxidase was obtained by this method.
Assuntos
Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Urato Oxidase/biossíntese , Alopurinol/farmacologia , Aspergillus flavus/efeitos dos fármacos , Sequência de Bases , Biotecnologia/métodos , Southern Blotting , Códon/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Resistência Microbiana a Medicamentos , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese , Plasmídeos , Regiões Promotoras Genéticas , Mapeamento por Restrição , Regiões Terminadoras Genéticas , Urato Oxidase/genéticaRESUMO
A transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.
Assuntos
Aspergillus flavus/genética , Transformação Genética/genética , Urato Oxidase/genética , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Southern Blotting , Clonagem Molecular , Teste de Complementação Genética , Mutação/genética , Nitrato Redutase , Nitrato Redutases/genética , Plasmídeos/genética , Mapeamento por Restrição , Urato Oxidase/metabolismo , Ácido Úrico/farmacologiaRESUMO
Batch processes for recombinant gene expression in prokaryotic systems should optimally comprise a growth phase with minimal promoter activity followed by a short phase favoring expression. The strong promoter of the tryptophan operon (Ptrp) gives high-level expression of recombinant proteins in E. coli. The inefficiency to control basal expression before induction is however a major obstacle towards the use of Ptrp, especially in the case of toxic proteins. To circumvent this problem, a novel E. coli strain has been generated. This mutant, named ICONE 200 (Improved Cell for Over and Non-leaky Expression), underwent replacement of tnaA, the tryptophanase encoding gene, with the trpR gene encoding the aporepressor of Ptrp. Detailed analysis of ICONE 200 showed that tryptophan, in addition to its natural role of Ptrp co-repressor, was able to induce trpR through the tryptophan-inducible tryptophanase promoter/operator. Consequently, Ptrp-dependent expression was efficiently repressed in the presence of tryptophan and was turned on, as in wild-type E. coli, as soon as tryptophan was exhausted from the medium. ICONE 200 has the capacity to express a wide range of proteins including toxic proteins such as HIV-1 protease and poliovirus 2B protein. ICONE 200 is a new host carrying stable, targeted, and marker-free genetic modifications and a candidate for large-scale applications.
Assuntos
Proteínas de Bactérias , Escherichia coli/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Repressoras/genética , Northern Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Protease de HIV/biossíntese , Protease de HIV/genética , Mutação , Proteínas Repressoras/metabolismo , Triptofano/metabolismo , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4(+), but not CD8(+), T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967-1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4(+) T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.