An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates

FEBS Journal - Tập 67 Số 1 - Trang 247-256 - 1976
Hugh R.B. Pelham1, Richard J. Jackson1
1Department of Biochemistry, University of Camdridge, Tennis Court Road, Cambridge, Great Britain, CB2 IQW

Tóm tắt

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 CaCl2 and micrococcal nuclease, and then excess ethyleneglycol‐bis(2‐aminoethylether)‐N,N′‐tetraacetic acid is added to chelate the Ca2+ 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.

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Thông tin
Thông tin xuất bản

FEBS Journal

Tập 67 Số 1

247-256

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