Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: Temperature plays a major role in protein yields

Soares, Carlos R.J.; Ueda, Eric; Oliveira, T.L.; Gomide, F.I.C.; Heller, S.R.; Bartolini, Paolo

doi:10.1016/j.jbiotec.2007.08.038

Cited by 26 publications

(37 citation statements)

References 34 publications

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“…8A), carried out first on the SP-Sepharose FF eluted fraction containing the two prolactin isoforms, showed a major peak of NG-hPRL with a relative molecular mass (M r ) of 22,888.8, i.e., different by only −0.04% from the calculated theoretical value (Soares et al, , 2008, as well as several minor peaks. The heterogeneous peak with the central form presenting a M r of 24,640, was apparently G-hPRL.…”

Section: Resultsmentioning

confidence: 94%

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: Resultsmentioning

confidence: 94%

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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“…Com exceção do IFNα2a desenvolvido durante esse estudo, as outras proteínas, hGH, hPRL e os antagonistas de prolactina G129R-hPRL e Δ1-9 G129R-hPRL já haviam sido produzidas no nosso laboratório (Oliveira, 1999;Soares, 2003;Soares, 2008, Heller, 2010 Nos estudos com o promotor  P L constitutivo, apesar da expressão ser teoricamente contínua, continuamos empregando o termo "ativação" para indicar o momento no qual a temperatura é elevada para o "início" da expressão. Embora em teoria esse termo "ativação" não faça muito sentido quando a expressão é constitutiva, na prática temperaturas mais baixas tendem a diminuir a ação do promotor  P L como relatado por Valdez-Cruz e colaboradores (2010).…”

Section: Discussionunclassified

“…Observou-se que em temperaturas iguais ou abaixo de 37 ºC a expressão de hGH é mínima, enquanto a 42 ºC é de 1,8 g hGH/mL/A 600 (Soares, 2008).…”

Section: Fernanda Dos Santos Arthuso Perezunclassified

“…Esse é um modelo de expressão bastante interessante, pois permite o funcionamento eficiente do promotor em temperaturas mais baixas de cultivo, o que aumenta a eficiência da expressão de proteínas como a hPRL, que se mostrou extremamente instável quando o cultivo bacteriano foi realizado com temperaturas superiores a 37 °C. Essa estratégia resultou em um aumento significativo na expressão da hPRL que passou para aproximadamente 1 µg hPRL/mL/A 600 (Soares, 2008).…”

Section: Fernanda Dos Santos Arthuso Perezunclassified

“…Optamos por não utilizar o plasmídeo do repressor pRK248cIts, cuja presença prejudicou a expressão da hPRL (Soares, 2008). …”

Section: Transformação Dos Plasmídeos Em Bactérias Escherichia Coliunclassified

See 2 more Smart Citations

Influência da temperatura de cultivo na expressão de proteínas recombinantes de interesse terapêutico no espaço periplásmico bacteriano, utilizando o promotor lambda PL

Perez¹

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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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Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: Temperature plays a major role in protein yields

Cited by 26 publications

References 34 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

Influência da temperatura de cultivo na expressão de proteínas recombinantes de interesse terapêutico no espaço periplásmico bacteriano, utilizando o promotor lambda PL

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

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