Please use this identifier to cite or link to this item:
|Title:||Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin||Authors:||Goh, Y.Y.
Quantitative LPS detection
|Issue Date:||2002||Citation:||Goh, Y.Y.,Frecer, V.,Ho, B.,Ding, J.L. (2002). Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin. Protein Engineering 15 (6) : 493-502. ScholarBank@NUS Repository.||Abstract:||Enhanced green fluorescent protein (EGFP) was selected as a signalling scaffold protein for design of a fluorescent biosensor for bacterial endotoxin [or lipopolysaccharide (LPS)]. Virtual mutagenesis was utilized to model EGFP variants containing binding sites for LPS and lipid A (LA), the bioactive component of LPS. Cationic amphipathic sequences of five alternating basic and hydrophobic residues were introduced to β-sheets located on the surface of EGFP barrel, in the vicinity of the chromophore. Computational methods were employed to predict binding affinity of Escherichia coli LA, to the models of virtual EGFP mutants. DNA mutant constructs of five predicted best binding EGFP variants were expressed in COS-1 cells. The EGFP-mutant proteins exhibited differential expression and variable degrees of fluorescence yield at 508 nm. The EGFP mutants showed a range of LA binding affinities that corresponded to the computational predictions. LPS/ LA binding to the mutants caused concentration-dependent fluorescence quenching. The EGFP mutant, G10 bearing LPS/LA amphipathic binding motif in the vicinity of the chromophore (YLSTQ200-204→KLKTK) captured LA with a dissociation constant of 8.5 μm. G10 yielded the highest attenuation of fluorescence intensity in the presence of LPS/ LA and demonstrated capability in fluorescence-mediated quantitative detection of LPS in endotoxin-contaminated samples. Thus, the EGFP mutant can form the basis of a novel fluorescent biosensor for bacterial endotoxin.||Source Title:||Protein Engineering||URI:||http://scholarbank.nus.edu.sg/handle/10635/101526||ISSN:||02692139|
|Appears in Collections:||Staff Publications|
Show full item record
Files in This Item:
There are no files associated with this item.
checked on Nov 9, 2019
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.