title: Construction of a novel glucose-sensing molecule based on a substrate-binding protein for intracellular sensing.
abstract: A novel transcriptional regulator responding to glucose was designed with a substrate-binding protein (SBP) as a probe towards intracellular sensing system for glucose in mammalian cells. A chimeric protein of an SBP for glucose (GBP) and a LacI-type regulator, LacI (SLCP(GL) ), was designed, constructed and characterized using Escherichia coli recombinant protein. We report that SLCP(GL) has a glucose-specific binding ability and an operator-sequence specific DNA-binding ability. The loss of its DNA-binding ability in the presence of glucose suggests a role as a transcriptional regulator in vitro. The glucose-dependent gene regulation function of SLCP(GL) in cells was investigated using mammalian cells co-transfected with SLCP(GL) and Lac operator-fused luciferase gene constructs. The luciferase activity of the transfected cells increased with the glucose concentration in the medium, showing that the expression of the luciferase gene is regulated by SLCP(GL) , which can dissociate from DNA in a glucose concentration-dependent manner. Therefore, we demonstrated that SLCP(GL) functions as a glucose-sensitive transcriptional regulator in mammalian cells. These results reveal the possibility of developing an SBP-based regulator as a probe of intracellular sensing and gene regulation system for mammalian cells in response to a desired ligands depending on the SBP ligand specificity.
authors.affiliation: International Center for Materials Nanoarchitectonics, National Institute for Materials Science, Namiki, Tsukuba, 305-0044 Ibaraki, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Sakaguchi-Mikami
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Taniguchi
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Yamazaki
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005947:Glucose / Q000032:analysis* / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D056968:Lac Repressors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D011994:Recombinant Proteins; D004247:DNA; D005947:Glucose