Optimization of an In Vitro Transcription/Translation System Based on Cell Lysate.

A system is described which permits the efficient synthesis of proteins at high temperature. It is based on the use of an unfractionated cell lysate (S30) from previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the DNA-alkylguanine-DNA-alkyl-transferase protein (OGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.