title: Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space.
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®). 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/A600(∼0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 °C up to 3 A600units 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.
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil.
authors.forename: Ana C S C
authors.initials: ACSC
authors.lastname: Menezes
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil.
authors.forename: Miriam F
authors.initials: MF
authors.lastname: Suzuki
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil.
authors.forename: João E
authors.initials: JE
authors.lastname: Oliveira
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil.
authors.forename: Maria T C P
authors.initials: MTCP
authors.lastname: Ribela
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil.
authors.forename: Isadora C
authors.initials: IC
authors.lastname: Furigo
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil.
authors.forename: José
authors.initials: J
authors.lastname: Donato
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bartolini
authors.affiliation: Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, São Paulo, Brazil. Electronic address: crsoares@ipen.br.
authors.forename: Carlos R J
authors.initials: CRJ
authors.lastname: Soares
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D020125:Mutation, Missense; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone
keywords: Escherichia coli; Growth hormone receptor antagonist; Lambda PL promoter; Periplasmic space