Protein Synthesis and Co-Translational Folding in Cell-Free Translation Systems

Abstract

The cell-free methodology for the synthesis of functionally active proteins is considered, and the so-called continuous cell-free translation and transcription-translation systems are described. The continuous cell-free systems for gene expression are based on the use of a porous barrier that retains the high-molecular-weight components of the protein-synthesizing machinery within a defined reaction compartment, and at the same time provides the continuous feeding with substrates (NTPs and amino acids) and the removal of reaction products. There are two versions of the continuous systems: the flow version (continuous-flow cell-free, or CFCF systems) and the dialysis version (continuous-exchange cell-free, or CECF systems). Both versions have been shown to provide a prolonged synthesis of proteins, as compared with standard (batch) cell-free systems, and correspondingly a significantly higher yield of proteins synthesized. The synthesis of fusion proteins and the direct expression of PCR products in cell-free systems are discussed as promising methodological approaches in a number of cases. Using the monitoring of polypeptide elongation in cell-free systems the evidence is presented that the folding of synthesized polypeptides into functional protein globules proceeds on ribosomes during translation (co-translational protein folding).