β galactosidase enzyme fragment complementation as a high-throughput screening protease technology
β galactosidase enzyme fragment complementation as a high-throughput screening protease technology
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DOI：
10.1177/1087057104264040
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发表时间：
2004-08-01
影响因子：
--
通讯作者：
Eglen, RM
中科院分区：
文献类型：
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作者：
Naqvi, T;Lim, A;Eglen, RM
作者描述了一种用于测量蛋白酶活性和检测抑制剂的同质高通量筛选 (FITS) 测定法。该测定包含含有蛋白酶选择性切割序列的环状 β-半乳糖苷酶 (β-gal) 酶供体肽 (ED)。环肽单独时是无活性的，但当蛋白酶裂解后线性化时，ED 与 β-gal 酶受体互补，形成活性 β-gal 酶。然后催化荧光或化学发光产物的形成，β-半乳糖转换提供高度放大的信号，从而提供高灵敏度的测定技术。为了证明该技术的实用性，开发了 EFC 测定法来测量 2、半胱天冬酶 3 和 β 分泌酶的活性。使用含有 caspase 3 底物序列 DEVD 的循环 ED，EFC 测定信号与 caspase 3 浓度呈线性关系。该测定非常灵敏，能够检测低皮克量的半胱天冬酶 3 的活性。对于 β 分泌酶 (BACE) EFC 测定，使用了含有淀粉样前体蛋白 (APP) 瑞典突变体裂解位点 SEVNLDAEFK 的循环 ED。与 caspase 3 测定类似，BACE 活性诱导的信号与酶浓度呈线性关系，并且高度灵敏，能够检测纳克量的 BACE。该测定也比市售的基于 FRET 的 BACE 活性测定更灵敏。结论是，EFC 蛋白酶测定是一种简单、灵活、灵敏的蛋白酶 HTS 技术。
The authors describe a homogeneous, high-throughput screening (FITS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic beta-galactosidase (beta-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with beta-gal enzyme acceptor forming active beta-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with beta-gal turnover providing a highly amplified signal, and thus an assay technology of high sensitivity. To demonstrate the utility of the technology, an EFC assay was developed to measure the activity of 2, caspase 3 and beta-secretase. Using a cyclic ED containing the caspase 3 substrate sequence, DEVD, the EFC assay signal was linear with respect to caspase 3 concentration. The assay was very sensitive, being able to detect activity at low picogram amounts of caspase 3. For the beta-secretase (BACE) EFC assay, a cyclic ED containing the Swedish mutant cleavage site of amyloid precursor protein (APP), SEVNLDAEFK, was used. In a similar fashion to the caspase 3 assay, the signal induced by BACE activity was linear with respect to enzyme concentration and was highly sensitive, being able to detect nanogram quantities of BACE. The assay was also more sensitive than a commercially available FRET-based assay of BACE activity. It is concluded that the EFC protease assay is a simple, flexible, and sensitive technology for HTS of proteases.