JOSÉ MARIA SOUZA
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Resumo IPEN-doc 07731 Utilization of reversed-phase high performance liquid chromatography (RP-HPLC) for the analysis of recombinant thyrotropin directly in CHO cell conditioned medium2001 - OLIVEIRA, J.E.; MENDONCA, F.; PERONI, C.N.; SOUZA, J.M.; BARTOLINI, P.; RIBELA, M.T.C.P.Resumo IPEN-doc 11011 Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells2005 - RIBELA, M.T.C.P.; CARVALHO, C.M.; HELLER, S.R.; LOUREIRO, R.F.; OLIVEIRA, J.E.; OZAKI, N.A.; PERONI, C.N.; SOARES, C.R.J.; SOUZA, J.M.; UEDA, E.K.; BARTOLINI, P.The synthesis and laboratory production of human growth hormone (hGH) and prolactin (hPRL) have been carried out in genetically modified E. coli, while those of thyroid stimulating hormone (hTSH) and of two analogs antagonists of hPRL (G129RhPRL and S179D-hPRL) in stably transfected CHO cells. Human follicle stimulating hormone (hFSH) and luteinizing hormone (hLH) have not been synthesized yet in our laboratory but their HPLC analytical methodologies are under development. For the purpose of studying and improving synthesis and bioreaction yields and, at the same time, planning and following all subsequent purification steps, novel RP-HPLC methods have been set up for each hormone. For hGH and hPRL, isocratic RP-HPLC methods have been developed that can qualitatively and quantitatively analyze the two hormones directly in osmotic shock fluids, already during fermentation. For hTSH, hFSH and hLH, RP-HPLC gradient elutions have been set for the analysis of these hormones in their purified form and in CHO conditioned medium. For these three glycoproteins hydrophobicities have been compared and the following order established: hLH>hTSH>hFSH. An analogous hydrophobicity index has been also determined for hPRL and its analogs, being G129R-hPRL>hPRL>S179D-hPRL. Still concerning hFSH, for the first time it has been possible to optimize RP-HPLC elution conditions that are able to preserve its undissociated heterodimeric structure. Thanks to this tool it was thus possible to carry out a comparative study on pituitary, urinary and CHO-derived hFSH preparations, revealing differences that are probably due to the carbohydrate moiety, as already observed for hTSH. Classical highperformance size exclusion chromatography (HPSEC) together with MALDI-TOF-MS analysis was also employed along with these studies, to complement physico-chemical characterization of our proteins of interest.Resumo IPEN-doc 06990 Hollow fiber bioreactors for hormone production in CHO cells2000 - SOARES, C.R.J.; PERONI, C.N.; MORGANTI, L.; AFFONSO, R.; ARKATEN, R.R.; SOUZA, J.M.; BARTOLINI, P.