Purification and Properties of the Polypeptide Chain Release Factor 1 (RF-1) from an Extreme Thermophile, Thermus thermophilus HB8

Abstract

The polypeptide chain release factor 1 (RF-1) has been purified from an extreme thermophile, Thermus thermophilus HB8. The purification procedure included steps of aqueous two-phase partition, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, Sephadex G-150, and CM-Sephadex. The preparation was more than 90% pure as judged by polyacrylamide gel electrophoresis. The specific activity was about 3.3 pmol of formyl-[³H] methionine released in 1 min at 25°C per μg of protein under the standard assay conditions using 4 pmol of the initiation complex and 1 nmol of UpApG. The molecular weight as determined by gel filtration on Sephadex G-150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58, 000 and 45, 000, respectively. As expected, the factor was extremely heat-stable, 50% of its activity remaining after incubation for 5min at 84°C. Several properties of the reaction catalyzed by RF-1 are also described.