Cell-free bioluminescent screening of translation inhibitors.
The possibility of creating new screening methods with a cell-free translation system has been demonstrated with a quantitative determination of diphtheria toxin and some antibiotics (puromycin, kanamycin and tetracycline) as examples. The approach proposed follows from the ability of various substances to inhibit protein synthesis. We used a wheat-germ cell-free translation system stabilized by freeze-drying in the presence of trehalose with the mRNA of the Ca(2+)-activated photoprotein obelin as a reporter template. This freeze-dried cell-free translation system allows prolonged storage of the detecting system before it is required, increases the reproducibility of the results and simplifies the application procedure. The obelin mRNA extends the sensitivity of the method owing to the high sensitivity of detection of the synthesized protein.
Branch of the Institute of Bioorganic Chemistry RAS, Pushchino, Moscow Region, Russia.
Gorokhovatsky AYu
L A
LA
Shaloiko
V S
VS
Bondar
E S
ES
Vysotski
E E
EE
Maximov
H
H
von Doehren
Y B
YB
Alakhov
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002474:Cell-Free System; D004167:Diphtheria Toxin / Q000494:pharmacology*; D007612:Kanamycin / Q000494:pharmacology; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000096:biosynthesis; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011691:Puromycin / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004167:Diphtheria Toxin; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; C010497:obelin; D011691:Puromycin; D007612:Kanamycin; D013752:Tetracycline