High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system.

Two modifications are introduced to convert the Escherichia coli cell-free extract ("S30") into a high efficiency system for coupled transcription/translation of exogenously added genes. (a) The ribosome fraction collected from the S30 by ultracentrifugation is used. It contains all the proteins necessary for gene expression but has lost the vast majority of soluble proteins that might interfere with purification and enzymatic activity of product formed. (b) Plasmids containing coding sequences to be expressed are not linearized thus enhancing their stability by avoiding their degradation. These two modifications not only improve protein synthesis in a static system but allow gene expression over 20-40 h in the continuous-flow cell-free system. Both prokaryotic and eukaryotic proteins have been synthesized in this system.