SELECTIVE INACTIVATION OF EUKARYOTIC BETA-GALACTOSIDASE IN ASSAYS FOR INHIBITORS OF HIV-1 TAT USING BACTERIAL BETA-GALACTOSIDASE AS A REPORTER ENZYME
SELECTIVE INACTIVATION OF EUKARYOTIC BETA-GALACTOSIDASE IN ASSAYS FOR INHIBITORS OF HIV-1 TAT USING BACTERIAL BETA-GALACTOSIDASE AS A REPORTER ENZYME
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DOI：
10.1006/abio.1993.1549
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发表时间：
1993-11-15
影响因子：
2.9
通讯作者：
DUTKO, FJ
中科院分区：
文献类型：
--
作者：
YOUNG, DC;KINGSLEY, SD;DUTKO, FJ
细菌β-半乳糖苷酶是用于研究真核启动子转录活性的几种报告酶之一。尽管它是最容易测定且最便宜的酶之一，但由于其灵敏度低，其使用受到限制，部分原因是许多真核细胞中β-半乳糖苷酶的内源水平。在这项研究中，我们比较了细菌和真核β-半乳糖苷酶的pH和盐需求以及热稳定性，以确定抑制真核细胞内源性β-半乳糖苷酶的条件，而不会对表达载体转染后产生的纯化细菌β-半乳糖苷酶或报道β-半乳糖苷酶的活性产生不利影响。 真核细胞。 50°C 热处理 1 小时，可使几种真核细胞系内源性 β-半乳糖苷酶活性灭活多达 40 倍，且不会对细菌 β-半乳糖苷酶活性产生不利影响。这种处理提高了该报告酶的敏感性，并允许开发一种快速且可量化的 HIV-1 tat 抑制剂筛选测定方法。
Bacterial β-galactosidase is one of several reporter enzymes used in studying the transcriptional activity of eukaryotic promoters. Although it is one of the easiest and least expensive enzymes to assay, its use has been limited because of its low sensitivity, which is due in part to endogenous levels of β-galactosidase in many eukaryotic cells. In this study, we compared the pH and salt requirements, as well as the heat stability, of bacterial and eukaryotic β-galactosidase in order to identify conditions which would inhibit the β-galactosidase enzyme endogenous to eukaryotic cells without adversely affecting the activity of either purified bacterial β-galactosidase or reporter β-galactosidase produced after transfection of expression vectors into eukaryotic cells. Heat treatment at 50°C for 1 h inactivated the β-galactosidase activity endogenous to several eukaryotic cell lines by as much as 40-fold without adversely affecting the activity of bacterial β-galactosidase. This treatment increased the sensitivity of this reporter enzyme and allowed the development of a rapid and quantifiable screening assay for HIV-1 tat inhibitors.