RESUMEN
A recombinant plasmid providing for the synthesis and secretion of the human atrial natriuretic peptide (hANP) as a C-terminal hybrid with the St. aureus protein A was constructed. The level of secretion of the chimeric proteins and their proteolytic stability were shown to depend upon the genotype of the recipient strains and the cultivation conditions. The hybrid proteins were purified by chromatography on IgG Sepharose. The presence of peptides corresponding to the hANP in the acid hydrolysates of the secreted and affinity-purified proteins was confirmed by the enzyme-linked immunoassay and analytical HPLC.
Asunto(s)
Factor Natriurético Atrial/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Factor Natriurético Atrial/química , Factor Natriurético Atrial/metabolismo , Secuencia de Bases , Humanos , Hidrólisis , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteína Estafilocócica A/genéticaRESUMEN
E. coli strains producing a hybrid protein containing human adrenocorticotropic hormone (ACTH) and protein A of S. aureus were obtained. The sequence coding for ACTG was obtained from the bovine one using oligonucleotide-directed mutagenesis. The ACTG gene was linked with the protein A gene and its derivatives by synthetic adaptors. It was shown that each of the constructed plasmids directed the synthesis of hybrid protein in E. coli. This protein was purified on IgG-Sepharose. Then ACTH was obtained by HPLC after specific hydrolysis. The amino acid composition of purified sample was determined.
Asunto(s)
Hormona Adrenocorticotrópica/genética , Escherichia coli/genética , Proteínas Recombinantes de Fusión/genética , Animales , Bovinos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Recombinante , Electroforesis en Gel de Poliacrilamida , Humanos , PlásmidosAsunto(s)
Hormona Adrenocorticotrópica/genética , Escherichia coli/genética , Proteínas Recombinantes de Fusión/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Regiones Promotoras Genéticas , Proteína Estafilocócica A/genéticaRESUMEN
E. coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S. aureus was obtained. The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors. Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene. The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively. It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E. coli. This protein was purified by the use of IgG-sepharose. The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture. Most of the synthesized protein is secreted into the periplasmic space.
Asunto(s)
Hormona Adrenocorticotrópica/genética , Escherichia coli/genética , Ingeniería Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN/genética , Datos de Secuencia Molecular , Plásmidos , Proopiomelanocortina/genética , Proteínas Recombinantes/genética , Proteína Estafilocócica A/genéticaAsunto(s)
Hormona Adrenocorticotrópica/biosíntesis , Escherichia coli/metabolismo , Biosíntesis de Péptidos , Hormona Adrenocorticotrópica/genética , Hormona Adrenocorticotrópica/metabolismo , Animales , Bovinos , ADN Bacteriano/análisis , Escherichia coli/genética , Expresión Génica , Péptidos/metabolismo , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/genéticaRESUMEN
The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.
