RESUMEN
F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-1/metabolismo , Chaperonas Moleculares/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , SolubilidadRESUMEN
Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Escherichia coli/genética , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/aislamiento & purificación , Genes Sintéticos , Humanos , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Expression E. coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter. The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance. The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.
Asunto(s)
Escherichia coli/genética , Expresión Génica , Genes Sintéticos , Interleucina-4/genética , Secuencia de Bases , Humanos , Interleucina-4/aislamiento & purificación , Interleucina-4/farmacología , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Linfocitos T/efectos de los fármacosRESUMEN
Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.
Asunto(s)
Bacillus subtilis/enzimología , GTP Ciclohidrolasa/genética , Clonación Molecular , ADN Bacteriano/genética , Electroforesis , Magnesio/metabolismo , Manganeso/metabolismo , Operón , Plásmidos , Biosíntesis de Proteínas , Mapeo Restrictivo , Riboflavina/genéticaRESUMEN
A modified H-phosphonate method was used to synthesize 32 oligodeoxyribonucleotides ranging in length from 23 to 28, which were enzymatically joined together to give the human interleukin 4 gene. The high degree of the oligonucleotide purity, achieved through the application of anion-exchange and reverse phase HPLC, ensures the high percentage of the desired sequence (about 75%) in the cloned DNA.
Asunto(s)
Genes Sintéticos , Genes , Interleucina-4/genética , Secuencia de Aminoácidos , Secuencia de Bases , Fenómenos Químicos , Química , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , OrganofosfonatosRESUMEN
The solid-phase phosphotriester method was used to synthesise 24 oligodeoxyribonucleotides, which were enzymatically joined together to give the human epidermal growth factor gene and its analogue containing Leu codon in position 21. The primary structure of the cloned genes were confirmed by the Maxam-Gilbert technique. It is demonstrated that the high purity degree of oligonucleotides allows to synthesise and clone genes without purification of intermediate fragments.
Asunto(s)
Clonación Molecular , Factor de Crecimiento Epidérmico/genética , Genes Sintéticos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/síntesis químicaRESUMEN
A recombinant plasmid has been constructed, which directs the synthesis of a hybrid protein, yeast repressible acid phosphatase [Val8]calcitonin, in yeast. The plasmid contains a truncated gene (pho5) acid phosphatase lacking 96 C-terminal amino acids replaced by the synthetic gene for human calcitonin and sequences required for the plasmid propagation in transformed yeast cells. A modified RIA method using immobilisation of protein extracts on solid supports was developed to monitor the expression of the hybrid protein. By use of this method, as well as by standard RIA of CNBr-cleaved protein extracts, synthesis of a calcitonin-related protein was detected in extracts of transformed strains grown under conditions inducing pho5 promoter.
Asunto(s)
Calcitonina/genética , Genes Sintéticos , Saccharomyces cerevisiae/genética , Valina , Secuencia de Aminoácidos , Calcitonina/biosíntesis , Humanos , Plásmidos , Proteínas Recombinantes/biosíntesisRESUMEN
Using two consecutive oligo(dT)-cellulose column chromatography steps, the total poly(A)RNA was isolated from the livers of rats injected with phenobarbital (PB) or 3-methylcholanthrene (MC). During translation of the PB-induced mRNA in the reticulocyte lysate cell-free protein-synthesizing system, a single polypeptide with an apparent molecular weight of 50,000 was synthesized which was specifically immunoprecipitated by antibodies to major PB-inducible cytochrome P-450 PB-3. In contrast, after completion of MC-mRNA translation, the antibodies to major MC-induced cytochrome MC-2 precipitated from the incubation mixture 4-5 polypeptides, of which the largest one with an apparent molecular weight of 58,000 corresponded to cytochrome P-450 MC-2. During sucrose density gradient centrifugation, the PB- and MS-mRNAs with sedimentation coefficients of about 18S and 20S, respectively, were precipitated.