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Construction of a Maltose Biosensor using the Thermotoga maritima Maltose Binding Protein
Author(s) -
Masson Laura,
Dattelbaum Jonathan D
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.665.17
Subject(s) - maltose binding protein , maltose , thermotoga maritima , chemistry , biosensor , periplasmic space , biochemistry , thermophile , circular dichroism , biophysics , escherichia coli , biology , fusion protein , recombinant dna , enzyme , gene
Thermotoga martima is a thermophilic, gram‐negative bacterium found primarily in hotsprings and hydrothermal vents. Its thermophilic characteristics mean that proteins found in this microbe should be extremely stable, even at very high temperatures, making them ideal for manipulation and biosensor design. The protein of interest for our project is MBP, or maltose binding protein, which is found in the periplasm of the bacteria and is responsible for binding maltose. Ultimately, we hope to design a Tm MBP biosensor that is stable, can bind molecules other than maltose, and will fluoresce when the target molecule is bound. MBP contains a region known as the binding pocket that allows the protein to recognize and bind the maltose molecule. Using site‐directed mutagenesis of residues in and around the binding pocket, we constructed six single cysteine mutants in Tm MBP. Proper folding of the mutants was monitored by circular dichroism as compared to the wild type Tm MBP. We covalently attached environmentally‐sensitive fluorophores (ABD, NBD, and acrylodan) such that when maltose is bound, the environment surrounding the fluorophore changes and causes a change in the fluorescence intensity, thus giving a quantitative indicator of whether or not maltose is bound to the protein. Using fluorescence intensity measurements, the dissociation constants for these mutants were determined to vary from 9–70 μM. A stable and efficient Tm MBP biosensor would be a quick and inexpensive way of testing samples for the presence of specific molecules, including those that may currently be difficult to detect.