Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein.
Journal Article (Journal Article)
We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 Angstrom resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 +/- 1 degrees C and 144 +/- 2 degrees C in the absence and presence of 1 mM glucose, respectively). A series of fluorescent conjugates was constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions predicted to be responsive to ligand-induced conformational changes based on the structure. These conjugates were screened to identify engineered tmGBPs that function as reagentless fluorescent glucose biosensors. The Y13C*Cy5 conjugate is bright, gives a large response to glucose over concentration ranges appropriate for in vivo monitoring of blood glucose levels (1-30 mM), and can be immobilized in an orientation-specific manner in microtiter plates to give a reversible response to glucose. The immobilized protein retains its response after long-term storage at room temperature.
Full Text
Duke Authors
Cited Authors
- Tian, Y; Cuneo, MJ; Changela, A; Höcker, B; Beese, LS; Hellinga, HW
Published Date
- October 2007
Published In
Volume / Issue
- 16 / 10
Start / End Page
- 2240 - 2250
PubMed ID
- 17766373
Pubmed Central ID
- PMC2204141
International Standard Serial Number (ISSN)
- 0961-8368
Digital Object Identifier (DOI)
- 10.1110/ps.072969407
Language
- eng
Conference Location
- United States