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Artigo IPEN-doc 22999 Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space2017 - MENEZES, ANA C.S.C.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; RIBELA, MARIA T.C.P.; FURIGO, ISADORA C.; DONATO JUNIOR, JOSE; BARTOLINI, PAOLO; SOARES, CARLOS R.J.The human growth hormone receptor antagonist G120R-hGH precludes dimerization of GH and prolactin receptors and consequently JAK/STAT signaling. Some modifications in this antagonist resulted in a drug specific for the GH receptor, called Pegvisomant (Somavert®). However, the original G120R-hGH is usually synthesized in bacterial cytoplasm as inclusion bodies, not being a commercial product. The present work describes the synthesis and characterization of G120R-hGH secreted into bacterial periplasm and obtained with a vector based on a constitutive lambda-PL promoter. This antagonist can be useful for studies aiming at investigating the effects of a simultaneous inhibition of GH and prolactin signaling, as a potential anti-tumoral or anti-diabetic compound. G120R-hGH, synthesized using the W3110 E. coli strain, showed a yield of 1.34 ± 0.24 mg/ml/A600 (~0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 C up to 3 A600 units and induction at 37 C, for 6 h, with final 4.3 ± 0.3 A600. A laboratory scale purification was carried out using three chromatographic steps with a total yield of 32%, reaching 98% purity. The obtained protein was characterized by SDS-PAGE, Western Blotting, Mass spectrometry, RP-HPLC, HPSEC and in vitro proliferation bioassay. The proliferation assay, based on Ba/F3- LLP cells, shows that G120R-hGH (100 ng/ml) significantly inhibited (64%) the proliferative action of hGH (1 ng/ml). This is the first time that G120R-hGH is synthesized in bacterial periplasmic space and therefore correctly folded, without the initial methionine. The reasons for a divergent efficacy for antagonizing hGH versus hPRL is currently unknown and deserves further investigation.Resumo IPEN-doc 21449 Expressão do antagonista de receptor do hormônio do crescimento humano G120R-hGH no espaço periplástico de Escherichia coli e sua caracterização físico-química e biológica2015 - MENEZES, ANA C. da S.C. de; SOARES, CARLOS R.J.