Abstract
Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFP(i)) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFP(i) proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFP(i) G10 and G12 to lipid A were established. The K(D) values of 7.16 x 10(-7) M for G10 and 8.15 x 10(-8) M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP(i) biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFP(i) specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFP(i) in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.