Continuous cell-free translation systems: history of the invention, reaction modes, and applications

The idea that in vitro protein synthesis should be performed under conditions of continuous inflow of feed and removal of waste, rather than in a test-tube with a fixed capacity, was first realized in cell-free translation systems with matrix-immobilized template polynucleotides in flow-through columns (solid-phase translation systems) [1, 2]. Later it was discovered that a cell-free translation mixture could be confined within a flow-through cell by an ultrafiltration membrane, without immobilization on a matrix; in this case the flow fed the system and removed products, but the components of protein-synthesizing machinery were retained in the liquid-phase reactor [3, 4]. At about the same time (1988-1989) a simplified version of the continuous cell-free system was devised: incubation of the translation mixture in a simple dialysis bag was proposed for replenishing substrates and removing low-molecular-weight products by diffusional exchange across the membrane during the protein synthesis reaction [5]. Thus, the continuous-flow cell-free (CFCF) technique and the continuous-exchange cell-free (CECF) method were brought into use as two main formats of long-lived, high-productive continuous systems for in vitro protein syntheses. The methodology was applied to the synthesis of proteins both with prokaryotic and eukaryoti