A Fractionated Reticulocyte Lysate System for Studies on Protein Synthesis Initiation Factors
The supplemented rabbit reticulocyte lysate is one of the most active cell-free translation systems known and has been extensively used over the last 20 yr or so for in vitro translation of endogenous (globin), as well as of heterologous mRNAs. In addition, it has regularly served as the starting material for the purification, identification, and indeed the characterization of some of the many protein factors required for eukaryotic protein synthesis.
KeywordsGlycerol Titration Sedimentation MgCl2 Fractionation
- 1.Clemens, M. J. (1984) Translation of eukaryotic messenger RNA in cell-free extracts, in Transcription and Translation: A Practical Approach (Hames, B. J. and Higgins, S. J., eds.), IRL Press, pp. 231–270.Google Scholar
- 9.Etchison, D. E., Hansen, J., Ehrenfeld, E., Edery, I., Sonenberg, N., Milburn, S. C., and Hershey, J. W. B. (1984) Demonstration zn vztro that eukaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells. J. Virol. 51, 832–837.PubMedGoogle Scholar
- 10.Etchison, D. E., Milburn, S. C., Edery, I., Sonenberg, N., and Hershey, J. W. B. (1982) Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-Dalton polypeptide associated with eukaryotic initiation factor 3 and a cap binding protein complex. J. Biol. Chem. 257, 14,806–14,810.PubMedGoogle Scholar
- 15.Liebig, H.-D., Ziegler, E., Yan, R., Hartmuth, K., Klump, H., Kowalski, H., Blaas, D., Sommergruber, W., Frasel, L., Lamphear, B., Rhoads, R., Kuechler, E., and Skern, T. (1993) Purification of two Picornaviral 2A proteinases interaction with eIF4G and influence on in vitro translation. Biochemistry 32, 7581–7588.PubMedCrossRefGoogle Scholar
- 16.Thomas, A. M., Scheper, G. C., Kleijn, M., De Boer, M., and Voorma, H. O. (1992) Dependence of the adenovirus tripartite leader on the eIF4G subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of eIF4G cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation. Eur. J. Biochem. 207, 471–477.PubMedCrossRefGoogle Scholar