Principles and possible applications of in vitro protein synthesis

To understand the key processes of cell-free in vitro protein synthesis system which is based on combined transcription/translation, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. And continuous supply of RNA is needed in order to account for its loss via degradation to achieve the synthesis of reasonable amount of A-FABP. Furthermore, the amount of synthesized A-FABP was elevated by the continuous exchange of reaction mixture. For easy and rapid elucidation of protein function, high-throughput screening systems for protein synthesis are required. And we evaluated the possibility of miniaturizing protein synthesis by using small-scale reactor, such as a microchip for micro-total analysis system. A-FABP synthesis was successfully achieved with microchip. We also demonstrated the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time. These results suggest that the integration of in vitro protein synthesis system and subsequent analysis on a microchip would be useful for examination of each optimal protein expression condition, will thus accelerate proteome research.