Artigo de peri??dico
Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space
Registro en:
1046-5928
131
10.1016/j.pep.2016.12.001
16.148
Autor
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.
Resumen
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. Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP) FAPESP: 12/24345-4