Assuntos
Toxicologia , Toxicologia , Biodiversidade , Venenos , Intoxicação , Toxinas Biológicas , Toxicologia , Bioquímica , Genética , Zoologia , BiodiversidadeRESUMO
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
Recombinant, fully bioactive, authentic human prolactin (aut-hPRL) has been synthesized in transformed Escherichia coli HB2151 bacteria in a soluble, non-glycosylated form, which is secreted into the bacterial periplasm. Use was made of a bacterial expression vector, containing tac promoter-controlled sequences for the translation enhancer from bacteriophage T7 gene 10, and for a cellulase leader peptide from Cellulomonas fimi joined to sequences coding for aut-hPRL. This vector was derived from a previously described vector containing sequences of an hPRL variant, tag-hPRL (containing a 12-amino-acid peptide tag at the N-terminal end), using site-specific mutagenesis to delete the tag sequence. SDS/PAGE, partial N-terminal amino acid sequence analysis, Western blot analysis and Nb2 lymphoma cell in vitro bioassay indicated correct processing of the hormone. Periplasmic secretion of aut-hPRL, as measured by immunoassay, was relatively low (approx. 0.08 microgram/ml per A600 unit), in contrast to that of tag-hPRL which was approximately 8-fold higher, apparently a consequence of the tag sequence. This is the first report describing periplasmic secretion of biologically active, authentic hPRL.
Assuntos
Escherichia coli/metabolismo , Periplasma/metabolismo , Prolactina/biossíntese , Sequência de Aminoácidos , Bacteriófago T7/genética , Western Blotting , Celulase/genética , Celulase/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Humanos , Linfoma , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prolactina/química , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Células Tumorais CultivadasRESUMO
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/genéticaRESUMO
The studied family showed the presence of four different types of hemoglobin. The family member who gave rise to this study (=propositus) presented Hb C and the hybrid Hb CG-phila. The propositus has three children, all of which have Hb AC; none of the family members showed any clinical symptoms. The investigation of the hemoglobin arose from the finding of target red cells in a blood test done during the pre-operatory examination for lower limb varicose vein stripping. The hybrid Hb CG-phila is due to two gene pairs, each of which with individual expression, determining the synthesis and the particular type subunits. The hybrid Hb CG-phila is formed by the combination velocity of the subunits alpha 2G-phila beta 2; therefore the proportion of the hybrid Hb CG-phila is lower than Hb G-phila and Hb C. The identification and molecular characterization of Hb G-phila showed the position alpha 2(68) Asn-->Lys beta 2 and Hb C showed alpha 2 beta 2(6) Glu-->Lys.
