Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space

Autor(es)

Menezes, Ana C. S. C. ; Suzuki, Miriam F. ; Oliveira, Joao E. ; Ribela, Maria T. C. P. ; Furigo, Isadora C. ; Donato, Jr., Jose ; Bartolini, Paolo ; Soares, Carlos R. J.

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 131, p. 91-100, MAR 2017.
• Citações Web of Science: 4

Resumo

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 (R)). 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. coil strain, showed a yield of 1.34 +/- 0.24 mu g/ml/A(600) (-0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 degrees C up to 3 A(600) units and induction at 37 degrees C, for 6 h, with final 4.3 +/- 0.3 A(600). 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. (C) 2016 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 12/24345-4 - Avaliação do potencial de antagonistas de prolactina para o tratamento da obesidade e do Diabetes mellitus 2
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular