A long-lived batch reaction system of cell-free protein synthesis

Anal Biochem. 1995 Apr 10;226(2):320-4. doi: 10.1006/abio.1995.1231.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cell-Free System / metabolism*
  • Globins / biosynthesis
  • Kinetics
  • Protein Biosynthesis* / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Transfer / metabolism
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Tetrahydrofolate Dehydrogenase / biosynthesis
  • Tobacco Mosaic Virus
  • Triticum
  • Viral Proteins / biosynthesis


  • RNA, Messenger
  • RNA, Viral
  • Viral Proteins
  • Adenosine Triphosphate
  • Globins
  • RNA, Transfer
  • Tetrahydrofolate Dehydrogenase