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Protein Engineering, Vol. 15, No. 6, 493-502, June 2002
© 2002 Oxford University Press

Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin

Yan Y. Goh1, Vladimir Frecer1,2, Bow Ho3 and Jeak L. Ding1,4

1 Department of Biological Sciences and 3 Department of Microbiology, National University of Singapore, 14, Science Drive 4, Singapore 117543, Singapore

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 ß-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.


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