title: Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space.
abstract: 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 λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A₆₀₀ units, a specific yield of 0.06-0.1 µg mPRL/(mL A₆₀₀), and a concentration of up to 2.2 µg/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].
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.forename: Fernanda S
authors.initials: FS
authors.lastname: Arthuso
authors.forename: João E
authors.initials: JE
authors.lastname: Oliveira
authors.forename: Nélio A J
authors.initials: NA
authors.lastname: Oliveira
authors.forename: Herbert R
authors.initials: HR
authors.lastname: Goulart
authors.forename: Marcos V N
authors.initials: MV
authors.lastname: Capone
authors.forename: Maria Teresa C P
authors.initials: MT
authors.lastname: Ribela
authors.forename: Paolo
authors.initials: P
authors.lastname: Bartolini
authors.forename: Carlos R J
authors.initials: CR
authors.lastname: Soares
mesh_terms: D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D019897:Periplasm / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
chemical_list: D011388:Prolactin