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(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 authentic mouse prolactin obtained in Escherichia coli periplasmic space

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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]
Total Authors: 9
[1] IPEN CNEN SP, Ctr Biotechnol, Inst Pesquisas Energet & Nucl, BR-05580000 Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: Biotechnology and Applied Biochemistry; v. 59, n. 3, p. 178-185, MAY-JUN 2012.
Web of Science Citations: 5

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)