Selective Inactivation of Eukaryotic β-Galactosidase in Assays for Inhibitors of HIV-1 TAT Using Bacterial β-Galactosidase as a Reporter Enzyme

Abstract

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.