A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria
A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria
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DOI：
10.1128/aem.68.12.6343-6352.2002
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发表时间：
2002-12
影响因子：
4.4
通讯作者：
Y. Goh;B. Ho;J. Ding
中科院分区：
文献类型：
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作者：
Y. Goh;B. Ho;J. Ding
摘要基于理性计算设计，对增强型绿色荧光蛋白（EGFP）进行定点突变，构建内毒素和革兰氏阴性菌荧光生物传感器。构建了带有一条（G10）或两条（G12）内毒素结合基序的EGFP突变体（EGFPi），并在大肠杆菌宿主中表达。EGFPi蛋白被纯化并测试其作为新型荧光生物传感器的功效。在有效去除E. coli裂解物中，建立了EGFPi G10和G12与脂质A的结合亲和力。G10的KD值为7.16 × 10−7 M，G12的KD值为8.15 × 10−8 M。随着在宽范围的pH和离子强度内保持高亲和力，脂质A/脂多糖与EGFP i生物传感器的结合可以测量为EGFP突变体的浓度依赖性荧光猝灭。EGFPi特异性标记革兰氏阴性菌，如E。大肠杆菌和铜绿假单胞菌，以及其他革兰氏阴性菌在污染的水采样的环境。EGFPi在检测游离内毒素和活革兰氏阴性细菌中的这种双重功能形成了开发新型荧光生物传感器的基础。
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 (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFPi 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 EGFPi G10 and G12 to lipid A were established. The KD values of 7.16 × 10−7 M for G10 and 8.15 × 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 EGFPi biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPi 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 EGFPi in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.