An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates

TLDR

The study presents a simple method to convert rabbit reticulocyte lysate into an mRNA‑dependent protein synthesis system. The lysate is preincubated with CaCl₂ and micrococcal nuclease, then Ca²⁺ is chelated with EGTA to inactivate the nuclease, yielding a clean translation system. The treated lysate shows negligible endogenous activity but recovers 75 % of the original activity with globin mRNA, exhibits high efficiency and sensitivity, retains functional tRNA, translates diverse mRNAs up to ~200 kDa without incomplete products, and can translate plant viral RNAs with heterologous tRNA.

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.