Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells
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2005
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CONFERENCE ON PROTEIN EXPRESSION IN ANIMAL CELLS, 7th
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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.
Como referenciar
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. Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells. In: CONFERENCE ON PROTEIN EXPRESSION IN ANIMAL CELLS, 7th, Sept. 18-22, 2005, Crete, Grecia. Abstract... Disponível em: http://repositorio.ipen.br/handle/123456789/19930. Acesso em: 14 Mar 2025.
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