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

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

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6 Scopus citations

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].

Original languageEnglish (US)
Pages (from-to)178-185
Number of pages8
JournalBiotechnology and Applied Biochemistry
Volume59
Issue number3
DOIs
StatePublished - May 1 2012

Keywords

  • Bioassay
  • Escherichia coli
  • HPLC
  • Mouse prolactin
  • Recombinant hormone

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