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An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice.

Hartenbach S, Daoud-El Baba M, Weber W, Fussenegger M - Nucleic Acids Res. (2007)

Bottom Line: Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology.ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types.Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland.

ABSTRACT
For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (P(ART)) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals.

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Western blot analysis of ARG2 expression in HEK-293T cells transfected with pSH91 (pSH91, PSV40-ARG2-pA) and cultivated for 60 h. Lane 1, lysate from HEK-293T transfected with pSH91 (pSH91, PSV40-ARG2-pA); lane 2, lysate from untransfected control cells. The 35 kDa band, indicative for the fusion protein ARG2 is shown with a black arrow. The loading control (tubulin-α, 57kDa) is indicated with an open arrow. Migration of molecular mass markers (kDa) is indicated on the left.
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Figure 5: Western blot analysis of ARG2 expression in HEK-293T cells transfected with pSH91 (pSH91, PSV40-ARG2-pA) and cultivated for 60 h. Lane 1, lysate from HEK-293T transfected with pSH91 (pSH91, PSV40-ARG2-pA); lane 2, lysate from untransfected control cells. The 35 kDa band, indicative for the fusion protein ARG2 is shown with a black arrow. The loading control (tubulin-α, 57kDa) is indicated with an open arrow. Migration of molecular mass markers (kDa) is indicated on the left.

Mentions: Since the combination of ART2 and PART1 provided the best combination of low leaky and maximum expression levels, we sought to further validate this system. Immortalized cell lines of human, rodent and monkey origin were co-transfected with pSH91 and pSH93. Sixty hours post transfection, cells exposed to 10 mg/l l-arginine displayed low SEAP expression, whereas 1 g/l l-arginine induced high-level production of the reporter gene (Table 2). Moreover, expression integrity of ARG2 in mammalian cells was confirmed by western blot analysis using HEK293-T transiently transfected with pSH91 (PSV40-ARG2-pA) (Figure 5).Figure 5.


An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice.

Hartenbach S, Daoud-El Baba M, Weber W, Fussenegger M - Nucleic Acids Res. (2007)

Western blot analysis of ARG2 expression in HEK-293T cells transfected with pSH91 (pSH91, PSV40-ARG2-pA) and cultivated for 60 h. Lane 1, lysate from HEK-293T transfected with pSH91 (pSH91, PSV40-ARG2-pA); lane 2, lysate from untransfected control cells. The 35 kDa band, indicative for the fusion protein ARG2 is shown with a black arrow. The loading control (tubulin-α, 57kDa) is indicated with an open arrow. Migration of molecular mass markers (kDa) is indicated on the left.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175317&req=5

Figure 5: Western blot analysis of ARG2 expression in HEK-293T cells transfected with pSH91 (pSH91, PSV40-ARG2-pA) and cultivated for 60 h. Lane 1, lysate from HEK-293T transfected with pSH91 (pSH91, PSV40-ARG2-pA); lane 2, lysate from untransfected control cells. The 35 kDa band, indicative for the fusion protein ARG2 is shown with a black arrow. The loading control (tubulin-α, 57kDa) is indicated with an open arrow. Migration of molecular mass markers (kDa) is indicated on the left.
Mentions: Since the combination of ART2 and PART1 provided the best combination of low leaky and maximum expression levels, we sought to further validate this system. Immortalized cell lines of human, rodent and monkey origin were co-transfected with pSH91 and pSH93. Sixty hours post transfection, cells exposed to 10 mg/l l-arginine displayed low SEAP expression, whereas 1 g/l l-arginine induced high-level production of the reporter gene (Table 2). Moreover, expression integrity of ARG2 in mammalian cells was confirmed by western blot analysis using HEK293-T transiently transfected with pSH91 (PSV40-ARG2-pA) (Figure 5).Figure 5.

Bottom Line: Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology.ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types.Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland.

ABSTRACT
For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (P(ART)) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals.

Show MeSH
Related in: MedlinePlus