Development and validation of an improved inducer-regulator protein complex in the pBRES-regulated expression system
| Autor: | Richard N. Harkins, Janet A. Meurer-Ogden, Konstantin Levitsky, Fang Jin, Paul Szymanski |
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| Rok vydání: | 2008 |
| Předmět: |
Transgene
Genetic Vectors Regulator Breast Neoplasms Transfection Mice Hormone Antagonists Progesterone receptor Oximes Genetics Tumor Cells Cultured Animals Humans Inducer Transgenes Enzyme inducer Estrenes Receptor Luciferases Molecular Biology DNA Primers biology Protein engineering Alkaline Phosphatase Small molecule Molecular biology Cell biology Protein Structure Tertiary Gene Expression Regulation Neoplastic Mifepristone biology.protein Mutagenesis Site-Directed Molecular Medicine Female Receptors Progesterone |
| Zdroj: | Human gene therapy. 19(11) |
| ISSN: | 1557-7422 |
| Popis: | Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES. Using modeling-guided protein engineering, compensatory mutations were installed at positions W755 and V729 (hPR numbering) in the ligand-binding pocket of the pBRES regulator protein (pBRES RP) to accommodate the new inducer and allow induction of transgene expression to levels previously seen with MFP. The improved inducer-pBRES RP complex was validated in vitro by monitoring the induction of luciferase, murine secreted alkaline phosphatase, and human interferon beta transgenes in mouse skeletal muscle cells. The engineered pBRES demonstrated low levels of transgene expression in the absence, and high expression levels in the presence, of the new BLX-913 inducer. Findings presented here allow induction of the pBRES-regulated gene expression system by a compound with markedly lower anti-hPR activity than MFP, the previously best characterized inducer. |
| Databáze: | OpenAIRE |
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