Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space
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2017
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Protein Expression and Purification
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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.
Como referenciar
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. Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space. Protein Expression and Purification, v. 131, p. 91-100, 2017. DOI: 10.1016/j.pep.2016.12.001. Disponível em: http://repositorio.ipen.br/handle/123456789/27151. Acesso em: 11 May 2024.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.