Engineering of ligand specificity of periplasmic binding protein for glucose sensing

Sakaguchi-Mikami, Akane; Taneoka, Atsushi; Yamoto, Rie; Ferri, Stefano; Sode, Koji

doi:10.1007/s10529-008-9712-7

Cited by 41 publications

(48 citation statements)

References 22 publications

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“…6). Previous studies have shown that the specificity and affinity of PBPs for a ligand can be modified by cavity mutagenesis (60)(61)(62). Given the sequence and structural homology between IbpA and RBPs, we sought to design a variant of IbpA in which the specificities for myo-inositol and D-ribose are switched (i.e., an IbpA mutant that binds ribose with an affinity higher than that for inositol).…”

Section: Discussionmentioning

confidence: 99%

myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Herrou

Crosson

2013

J Bacteriol

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Section: Discussionmentioning

confidence: 99%

myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Herrou

Crosson

2013

J Bacteriol

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“…GBP [9] and MBP [10] from E. coli native forms (or wild type, WT) bind their respective ligands with high affinity; the K D values for GBP/G and MBP/M are 0.2 μmol L −1 and 3.5 μmol L −1 , respectively. Because both proteins contain tryptophan residues, they autofluoresce.…”

Section: Transduction Mechanismsmentioning

confidence: 99%

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

et al. 2012

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There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.

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“…Site-directed mutagenesis for the construction of SLCP GL mutants with a lower affinity for glucose (SLCP GmL ) was carried out on pC-SLCP GL by QuikChange, as previously described (Sakaguchi-Mikami et al, 2008).…”

Section: Genetic Manipulationsmentioning

confidence: 99%

“…The cells were resuspended in PBS. The cell lysate was obtained by sonication and then loaded onto a Ni-NTA column (Qiagen, Hilden, Germany); SLCP GL was purified as described previously (Sakaguchi-Mikami et al, 2008). The expression and purification levels of the SLCP GL were determined by SDS-PAGE.…”

Section: E Coli Recombinant Production Of Slcp Glmentioning

confidence: 99%

Construction of a novel glucose‐sensing molecule based on a substrate‐binding protein for intracellular sensing

Sakaguchi-Mikami

Taniguchi

Sode

et al. 2010

Biotech & Bioengineering

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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.

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Engineering of ligand specificity of periplasmic binding protein for glucose sensing

Cited by 41 publications

References 22 publications

myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

Construction of a novel glucose‐sensing molecule based on a substrate‐binding protein for intracellular sensing

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