Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space

Autor(es)

Suzuki, Miriam F. ^[1] ; Arthuso, Fernanda S. ^[1] ; Oliveira, Joao E. ^[1] ; Oliveira, Nelio A. J. ^[1] ; Goulart, Herbert R. ^[1] ; Capone, Marcos V. N. ^[1] ; Ribela, Maria Teresa C. P. ^[1] ; Bartolini, Paolo ^[1] ; Soares, Carlos R. J. ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN SP, Ctr Biotechnol, Inst Pesquisas Energet & Nucl, BR-05580000 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biotechnology and Applied Biochemistry; v. 59, n. 3, p. 178-185, MAY-JUN 2012.
• Citações Web of Science: 5

Resumo

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage ? (?PL). Fermentation in a pilot bioreactor was carried out at 30 degrees C, with 6 H of induction at 37 degrees C, reaching an optical density of 23 A600 units, a specific yield of 0.060.1 mu g mPRL/(mL A600), and a concentration of up to 2.2 mu g/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL {[}World Health Organization (WHO)-97/714]. (AU)

• Processo FAPESP: 07/59540-3 - Síntese e caracterização de prolactina de camundongos (mPRL) e de seu análogo (S177D-mPRL) em Escherichia coli
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular