TY - JOUR
T1 - Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space
AU - Suzuki, Miriam F.
AU - Arthuso, Fernanda S.
AU - Oliveira, Jõao E.
AU - Oliveira, Nélio A.J.
AU - Goulart, Herbert R.
AU - Capone, Marcos V.N.
AU - Ribela, Maria Teresa C.P.
AU - Bartolini, Paolo
AU - Soares, Carlos R.J.
PY - 2012/5/1
Y1 - 2012/5/1
N2 - 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].
AB - 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].
KW - Bioassay
KW - Escherichia coli
KW - HPLC
KW - Mouse prolactin
KW - Recombinant hormone
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U2 - 10.1002/bab.1008
DO - 10.1002/bab.1008
M3 - Article
C2 - 23586827
AN - SCOPUS:84862514254
SN - 0885-4513
VL - 59
SP - 178
EP - 185
JO - Biotechnology and Applied Biochemistry
JF - Biotechnology and Applied Biochemistry
IS - 3
ER -
