An In Vitro Assay to Screen for Translation Inhibitors

• Published in: Journal of microbiology and biotechnology Vol. 16; no. 10; pp. 1646 - 1649
• Main Authors: Song, Chin-Hee, Paik, Hyoung-Rok, Seong, Chi-Nam, Choi, Sang-Ki
• Format: Journal Article
• Language: Korean
• Published: 2006
• Subjects: In vitro translation; inhibitor; Streptomyces spp; luciferase; protein synthesis
• ISSN: 1017-7825; 1738-8872

Protein synthesis is the ultimate outcome of gene expression which, in turn, is regulated by several translation factors. We attempted to identify substances that can inhibit the translation process in vitro when the outcome protein is luciferase. To this end, we developed a sensitive cell-free protein synthesis assay using luciferase as the reporter. The synthesis of luciferase increased proportionately as mRNA was added to a $15-{\mu}l$reaction medium in concentrations raging from 5 ng to 500 ng. The maximum amount of luciferase was synthesized when the media were incubated at $25^{\circ}C$ for 40 min. The concentration of each compound that inhibited luciferase production by 50% ($IC_{50}$) was calculated. Hygromycin, puromycin, and cycloheximide yielded an $IC_{50}$ of 0.008, 0.8, and $0.7{\mu}g/ml$, respectively. A filtrate of Streptomyces spp. isolates inhibited protein synthesis up to S-fold when added to the in vitro translation assay mixture.