ERIC KINNOSUKE MARTINS UEDA
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Artigo IPEN-doc 11417 Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in chinese hamster ovary (CHO) cells2006 - SOARES, C.R.J.; GLEZER, A.; OKAZAKI, K.; UEDA, E.K.M.; HELLER, S.R.; WALKER, A.M.; GOFFIN, V.; BARTOLINI, P.The synthesis, puriWcation and characterization of G129R–hPRL and S179D–hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the Wrst time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 g/106 cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the speciWc denaturation, refolding and puriWcation conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step puriWcation process and included SDS–PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-Xight mass spectral (MALDI–TOF–MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP–HPLC). This last technique revealed a considerable diVerence in hydrophobicity due to a single amino acid substitution, with S179D–hPRL less (tRR D 0.85 § 0.010) and G129R–hPRL more (tRR D 1.10§ 0.013) hydrophobic than hPRL, where tRR is the relative retention time. The biological characterization was based on further reWnement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 £ 10¡3 for S179D–hPRL and 70£ 10¡5 for G129R–hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting eVects of hPRL in vivo in animal models of breast and prostate cancer.