Skip to main content
Plant Physiology logoLink to Plant Physiology
. 1980 Oct;66(4):584–587. doi: 10.1104/pp.66.4.584

Preparation and Characterization of a Highly Active Cell-free Protein-synthesizing System from Dry Pea Primary Axes 1

Willy J Peumans 1,2, Albert R Carlier 1, Bernard M Delaey 1
PMCID: PMC440683  PMID: 16661482

Abstract

A highly active and reliable cell-free protein-synthesizing system from dry pea primary axes has been developed. The system has been optimized with respect to both overall activity and translational fidelity. Under optimal conditions, the system incorporates nearly 1,000 picomoles leucine per 50 microliters incubation mixture. At limiting tobacco mosaic virus RNA concentrations, its efficiency exceeds 1,000 picomoles leucine per picomole template molecules. The maximal size of the polypeptides coded for by tobacco mosaic virus RNA goes up to 180,000. A procedure has been worked out to reduce the endogenous template activity of the system. the dramatic increase of the activity of this pea cell-free system as compared to previous attempts can be explained by the use of Cl-free buffers.

Full text

PDF
584

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  2. Carlier A. R., Peumans W. J. The rye embryo system as an alternative to the wheat system for protein synthesis in vitro. Biochim Biophys Acta. 1976 Nov 1;447(4):436–444. doi: 10.1016/0005-2787(76)90081-2. [DOI] [PubMed] [Google Scholar]
  3. Gordon J. I., Deeley R. G., Burns A. T., Paterson B. M., Christmann J. L., Goldberger R. F. In vitro translation of avian vitellogenin messenger RNA. J Biol Chem. 1977 Nov 25;252(22):8320–8327. [PubMed] [Google Scholar]
  4. Hickey E. D., Weber L. A., Baglioni C. Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability. Biochem Biophys Res Commun. 1978 Jan 30;80(2):377–383. doi: 10.1016/0006-291x(78)90687-3. [DOI] [PubMed] [Google Scholar]
  5. Hunter A. R., Farrell P. J., Jackson R. J., Hunt T. The role of polyamines in cell-free protein synthesis in the wheat-germ system. Eur J Biochem. 1977 May 2;75(1):149–157. doi: 10.1111/j.1432-1033.1977.tb11512.x. [DOI] [PubMed] [Google Scholar]
  6. Hunter T. R., Hunt T., Knowland J., Zimmern D. Messenger RNA for the coat protein of tobacco mosaic virus. Nature. 1976 Apr 29;260(5554):759–764. doi: 10.1038/260759a0. [DOI] [PubMed] [Google Scholar]
  7. JOHNSTON F. B., STERN H. Mass isolation of viable wheat embryos. Nature. 1957 Jan 19;179(4551):160–161. doi: 10.1038/179160b0. [DOI] [PubMed] [Google Scholar]
  8. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  9. Marcus A., Efron D., Weeks D. P. The wheat embryo cell-free system. Methods Enzymol. 1974;30:749–754. doi: 10.1016/0076-6879(74)30073-0. [DOI] [PubMed] [Google Scholar]
  10. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  11. Roberts B. E., Paterson B. M. 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 U S A. 1973 Aug;70(8):2330–2334. doi: 10.1073/pnas.70.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rychlik W., Zagórski W. Protein synthesizing system from wheat germ: efficient translation of synthetic and natural messages. Acta Biochim Pol. 1978;25(2):129–146. [PubMed] [Google Scholar]
  13. Zagorski W. Preparation and characteristics of a wheat embryo cell-free extract active in the synthesis of high molecular weight viral polypeptides. Anal Biochem. 1978 Jul 1;87(2):316–333. doi: 10.1016/0003-2697(78)90681-4. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES