Obelin mRNA--a new tool for studies of translation in cell-free systems.
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.
Matveev
S V
SV
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Illarionov
B A
BA
Vysotski
E S
ES
Bondaŕ
V S
VS
Markova
S V
SV
Alakhov
Y B
YB
D002474:Cell-Free System; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D012333:RNA, Messenger; C010497:obelin