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In Vitro Protein Synthesis

  • Jayne A. Matthews
  • Raymond A. McKee
Protocol
  • 713 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The technique of in vitro protein synthesis involves the translation of messenger RNA (mRNA) in a cell-free extract to yield polypeptide products. Both prokaryotic and eukaryotic systems have been developed, but for this experiment only a eukaryotic system will be considered. In 1973, Roberts and Paterson (3) demonstrated that a cell-free extract of wheat germ would faithfully translate rabbit globin mRNA and tobacco mosaic virus RNA to yield full-length polypeptide products. Other systems have also been developed, one of the most popular being the rabbit reticulocyte lysate developed by Pelham and Jackson (2) in 1976. Both wheat germ and reticulocyte lysates are used extensively, and both have advantages and disadvantages (Table 1), but the choice of system often depends on personal preference. For the experiment outlined in this chapter, the wheat germ lysate is used because it is easy to prepare and the starting material is readily available.

Keywords

Wheat Germ Magnesium Concentration Creatine Phosphokinase Amino Acid Solution Magnesium Acetate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Paterson, B.M., Roberts, B.E., and Kuff, E.L. (1977) Structural gene identification and mapping by DNA, mRNA hybrid-arrested cell-free translation. Proc. Natl. Acad. Sci. USA 74, 4370–4374.PubMedCrossRefGoogle Scholar
  2. 2.
    Pelham, H.R.B. and Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysate. Eur.J. Biochem. 67, 247–256.PubMedCrossRefGoogle Scholar
  3. 3.
    Roberts, B.E. and Paterson, B.M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc. Natl. Acad. Sci. USA 70, 2330–2334.PubMedCrossRefGoogle Scholar
  4. 4.
    Spindler, S., Siebert, P., Coffman, F., and Jurnak, F. (1984) Isolation of biological active mRNA. Biochem. Education 12, 22–25.CrossRefGoogle Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • Jayne A. Matthews
    • 1
  • Raymond A. McKee
    • 2
  1. 1.Department of Clinical Chemistry, Wolfson Research LaboratoriesQueen Elizabeth Medical CentreBirminghamUK
  2. 2.Department of BotanyUniversity of LeicesterLeicesterUK

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