RESUMO
A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.
Assuntos
Escherichia coli/genética , Hormônio do Crescimento Humano/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Clonagem Molecular , Hormônio do Crescimento Humano/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/análise , Escherichia coli/química , Escherichia coli/genética , Hormônio do Crescimento Humano/genética , Humanos , Pressão Osmótica , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaAssuntos
Hipergamaglobulinemia/genética , Imunoglobulina M/sangue , Síndromes de Imunodeficiência/genética , Transtornos Linfoproliferativos/genética , Cromossomo X , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Feminino , Ligação Genética , Marcadores Genéticos , Humanos , Hipergamaglobulinemia/complicações , Síndromes de Imunodeficiência/complicações , Transtornos Linfoproliferativos/complicações , MasculinoRESUMO
19 probes for CpG islands, mapping to Xq28, have been used as probes to construct a physical map of genes of this band of the human X chromosome. A total of 22 CpG islands have been precisely mapped in respect to known loci along the 9-10 Mb of Xq28. The fine mapping of such a large number of CpG islands has demonstrated that also in gene rich Giemsa light bands, like Xq28, gene distribution is non uniform: the CpG islands are clustered in the distal portion of the band in a 2 Mb region between the G6PD gene and the DXS15 locus. Moreover, 16 CpG islands were found between the G6PD and the RCP/GCP genes, a region of DNA of only about 300 kb. If this structural organization has a biological function it has yet to be determined. However, the isolation of large genomic regions enriched in gene sequences and the availability of cosmid or YAC contigs will provide the means to test the significance of such gene organization, as well as the material for large sequencing projects and gene search, for the identification of candidate genes for inherited disorders mapped to Xq28 and for comparative mapping.