Cell-free translation continuous flow system

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

Fig. 4. Massive production of green fluorescent protein (GFP) by the continuous-flow cell-free method. Sodium dodecyl sulfide-polyacrylamide gel electrophoresis analysis of GFP produced during 14 d of reaction. mRNA produced by transcription of circular plasmid of Ehime University (pEU) was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1 -pL aliquot of the mixture was run on the gel and protein bands were stained with Coomassie Brilliant Blue. The arrow shows GFP and st designates an authentic GFP band (0.5 pg).

A cell-free extract (S30) from E. coli K12 has been developed as an efficient coupled transcription/translation system which performs protein synthesis in vitro from the genes cloned in plasmids under the T7, T3, or SP6 promoter. Capping of the mRNA for eukaryotic proteins is apparently unnecessary with the S30 system. The coupled transcription/translation system, which was originally developed as the S3 0 translation system (122), can be used in a batchwise or continuous-flow mode (123,124). Use of the ribosome fraction collected from the S30 extracts is reported to improve the yield and efficacy further and more advantageously with nonlinearized plasmids than with linearized plasmids (125). 

---