A long-lived batch reaction system of cell-free protein synthesis.
Several reaction conditions of cell-free protein synthesis such as temperatures, buffers, tRNAs, and creatine phosphate were intensively investigated and optimized to prolong protein synthesis and make it more efficiently in a batch system. As a result of these modifications, the protein synthesis reaction continued for 10 h so that about 30 micrograms of dihydrofolate reductase (DHFR) protein derived from Escherichia coli was synthesized in 1 ml of reaction mixture. In this improved system, translational reactions of other mRNAs such as rabbit beta-globin, Xenopus beta-globin, and tobacco mosaic virus RNA also continued for about 10 h. In addition, protein synthesis directed by uncapped dhfr mRNA containing a viral cap-independent translation initiation-mediating sequence continued for 10 h, resulting in the synthesis of 18 micrograms of DHFR protein per milliliter of reaction mixture.
Kawarasaki
Y
Y
Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Japan.
Kawai
T
T
Nakano
H
H
Yamane
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism*; D005914:Globins / Q000096:biosynthesis; D007700:Kinetics; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis; D014027:Tobacco Mosaic Virus; D014908:Triticum; D014764:Viral Proteins / Q000096:biosynthesis
D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D005914:Globins; D012343:RNA, Transfer; D013762:Tetrahydrofolate Dehydrogenase