Matveev S V, Illarionov B A, Vysotski E S, Bondaŕ V S, Markova S V, Alakhov Y B
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Anal Biochem. 1995 Oct 10;231(1):34-9. doi: 10.1006/abio.1995.1499.
Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system. Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific activity was 3.6 +/- 0.4 x 10(15)photons per mg of the in vitro synthesized obelin (k=6.9s(-1)). The luminescence of the obelin was in a good correlation with the protein concentration calculated by the incorporation of [14C]Leu. The determination of the amount of de novo synthesized obelin based on measurement of its luminescence is one-thousand times more sensitive than the approach based on the incorporation of labeled amino acid. Thus, obelin mRNA has some advantages for evaluating the efficiency of cell-free translation when compared with standard methods.
