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， ... More
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 μg/ml/A (∼0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 °C up to 3 A units and induction at 37 °C， for 6 h， with final 4.3 ± 0.3 A. 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.
