[Stabilized reaction mixture for in vitro mRNA translation].
Here we describe a method for obtaining a ready-to-use stabilized reaction mixture for in vitro translation of mRNA. We also demonstrate the stabilization of a complete translation mixture containing wheat germ extract, amino acids, ATP, GTP, creatine phosphate, creatine kinase, and the reaction buffer by lyophilization in the presence of various sugars. The greatest stabilizing effect is achieved by supplementing the mixture with 10% (mass/volume) trehalose, which is also a unique translation activator, enhancing the translation of various mRNAs. A lyophilized complete translation mixture containing trehalose can be stored at 4-8 degrees C for several months without losing its activity. The mixture can be easily reconstituted by adding an aqueous mRNA solution and retains the potential for reproducible functioning. This allows the employment of such a cell-free translation system for analytical screening of a broad spectrum of compounds inhibiting translation at various stages.
Shaloĭko
L A
LA
Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia.
Gorokhovatskiĭ
A Iu
AIu
Maksimov
E E
EE
Alakhov
Iu B
IuB
D000255:Adenosine Triphosphate / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D002021:Buffers; D003402:Creatine Kinase / Q000737:chemistry; D005612:Freeze Drying; D006160:Guanosine Triphosphate / Q000737:chemistry; D008164:Luminescent Proteins / Q000138:chemical synthesis / Q000235:genetics; D010725:Phosphocreatine / Q000737:chemistry; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D014199:Trehalose / Q000737:chemistry*; D014908:Triticum / Q000737:chemistry
D000596:Amino Acids; D002021:Buffers; D008164:Luminescent Proteins; D012333:RNA, Messenger; C010497:obelin; D010725:Phosphocreatine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014199:Trehalose; D003402:Creatine Kinase