Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells

Soares, Carlos R.J.; Glezer, Andréa; Okazaki, Kazuichi; Ueda, Eric; Heller, S.R.; Walker, AM; Goffin, Vincent; Bartolini, Paolo

doi:10.1016/j.pep.2006.04.013

Cited by 22 publications

(39 citation statements)

References 51 publications

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“…MALDI-TOF-MS, carried out on the heterogeneous product, allowed identification and relative molecular mass determinations for NG-hPRL and G-hPRL confirming previously reported values (Price et al, 1995;Soares et al, 2006). Purified G-hPRL indicated the presence of different molecular isoforms in approximately the same range of those (M r 24,864-25,588) that were determined by Price et al (1995) via electrospray ionization mass spectroscopy for recombinant C127 cell-derived G-hPRL (Genzyme).…”

Section: Discussionsupporting

confidence: 80%

“…The G-hPRL present in conditioned culture medium was purified using a method described by Soares et al (2006). A two-step purification process was used: SP-Sepharose Fast Flow followed by a size-exclusion chromatography employing HPSEC for preparative purposes.…”

Section: G-hprl Purificationmentioning

confidence: 99%

“…Purified G-hPRL was then characterized by RP-HPLC and HPSEC, Western blotting, MALDI-TOF-MS and two in vitro bioassays. The first is the classical heterologous Nb2-rat lymphoma-cell-proliferation assay, while the second is the homologous Ba/F3-LLP assay, based on murine pro-B cells containing the sequence encoding the human PRL receptor (Tanaka et al, 1980;Soares et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

Heller

Goulart

Arthuso

et al. 2010

Journal of Biotechnology

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Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

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Section: Discussionsupporting

confidence: 80%

Section: G-hprl Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

Heller

Goulart

Arthuso

et al. 2010

Journal of Biotechnology

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“…Second, even polyclonal antibodies against PRL recognize S179D PRL as somewhat different from unmodified PRL (Chen et al, 1998). Third, S179D PRL has a number of altered physical properties which include, a decrease in hydrophobicity consistent with the addition of more than one negative charge versus the unmodified hormone (Soares et al,2006), an increased ability to bind heparin, and an inability to bind to nickel (unpublished data). These latter two determinations were exactly the opposite of what one would have predicted from an additional negative charge.…”

Section: Conformation Of S179d Prlmentioning

confidence: 95%

“…However, when used alone and at high concentrations in cells that cannot activate alternate splicing of the receptor (see later), it shows some agonist activity, with about a two log increase in the amount required for equivalent activity to the unmodified hormone (Bernichtein et al, 2001;Soares et al, 2006). However, there is no circumstance in vivo when unmodified PRL is absent.…”

Section: S179d Prl Inhibits Cell Proliferationmentioning

confidence: 99%

S179D prolactin: Antagonistic agony!

Walker

2007

Molecular and Cellular Endocrinology

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The aims of this review are threefold: First, to collate what is known about the production and activities of phosphorylated prolactin (PRL), the latter largely, but not exclusively, as illustrated through the use of the molecular mimic, S179D PRL; second, to apply this and related knowledge to produce an updated model of prolactin-receptor interactions that may apply to other members of this cytokine super-family; and third, to promote a shift in the current paradigm for the development of clinically important growth antagonists. This third aim explains the title since, based on results with S179D PRL, it is proposed that agents which signal to antagonistic ends may be better therapeutics than pure antagonists -hence antagonistic agony. Since S179D PRL is not a pure antagonist, we have proposed the term selective prolactin receptor modulator (SPeRM) for this and like molecules.

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

Suzuki

Arthuso

Oliveira

et al. 2012

Biotech and App Biochem

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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].

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Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells

Cited by 22 publications

References 51 publications

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

S179D prolactin: Antagonistic agony!

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

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