Requirement of continuous transcription for the synthesis of sufficient amounts of protein by a cell-free rapid translation system

To understand the key processes of cell-free protein synthesis, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined under various conditions. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. However, synthesis of A-FABP was not observed when an RNA fragment corresponding to the open reading frame of A-FABP was used in the reaction instead of the expression vector. Northern analysis revealed that the RNA that was added to the reaction mixture promptly underwent degradation. On the contrary, when the expression vector of A-FABP was employed, a strong RNA signal was observed over the entire incubation period. Thus, a continuous supply of RNA is needed in order to account for its loss via degradation to achieve the synthesis of reasonable amounts of A-FABP. Furthermore, the effect of continuous exchange of reaction mixture was also evaluated by measurement of the amount of synthesized A-FABP. (C) 2002 Elsevier Science (USA).