A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease
A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease
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DOI：
10.1073/pnas.062171599
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发表时间：
2002-05-14
影响因子：
11.1
通讯作者：
Scolnick, EM
中科院分区：
文献类型：
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作者：
Cummings, RT;Salowe, SP;Scolnick, EM
建立了荧光共振能量转移法测定炭疽杆菌致死因子（LF）蛋白酶活性的方法。合成了基于已知LF蛋白酶底物MEK 1的荧光16-mer肽，并发现其在预期位点被酶切割。将这项工作扩展到荧光19聚体肽，部分来源于已知LF蛋白酶靶标的共有序列，产生了更好的底物，切割效率提高了约100倍。在树脂上进一步修饰该肽序列以掺入供体/猝灭剂对，从而产生用于基于荧光共振能量转移的外观测定的底物。所有肽以相似的速率裂解，在100%转换时信号/背景范围为9-16。选择其中一种底物（表示为（Cou）Consensus（K（QSY-35）GG）-NH 2）进行额外的试验优化。开发了仅需要低纳摩尔水平的酶的基于板的测定用于筛选和抑制剂表征。
A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9-16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH2, was selected for additional assay optimization. A plate-based assay requiring only low nano-molar levels of enzyme was developed for screening and inhibitor characterization.