Abstract
A simple method is described for converting a standard rabbit reticulocyte cell‐free extract (lysate) into an mRNA‐dependent protein synthesis system. The lysate is preincubated with CaCl 2 and micrococcal nuclease, and then excess ethyleneglycol‐bis(2‐aminoethylether)‐N,N′‐tetraacetic acid is added to chelate the Ca 2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full‐sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.
Keywords
ReticulocyteNucleaseMessenger RNALysisProtein biosynthesisHeterologousTranslation (biology)Transfer RNABiochemistryBiologyRNATobacco mosaic virusMicrococcal nucleaseMolecular biologyGlobinChemistryVirusEnzymeVirologyGene
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Publication Info
- Year
- 1976
- Type
- article
- Volume
- 67
- Issue
- 1
- Pages
- 247-256
- Citations
- 3963
- Access
- Closed
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Cite This
Hugh R.B. Pelham,
Richard J. Jackson
(1976).
An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates.
European Journal of Biochemistry
, 67
(1)
, 247-256.
https://doi.org/10.1111/j.1432-1033.1976.tb10656.x
Identifiers
- DOI
- 10.1111/j.1432-1033.1976.tb10656.x