Advanced search
Start date
Betweenand
(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

Full text
Author(s):
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.
Total Authors: 8
Document type: Journal article
Source: Protein Expression and Purification; v. 131, p. 91-100, MAR 2017.
Web of Science Citations: 4
Abstract

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)

FAPESP's process: 12/24345-4 - Evaluation of the potential of prolactin antagonists to the treatment of obesity and Diabetes mellitus 2
Grantee:Carlos Roberto Jorge Soares
Support type: Regular Research Grants