Synthesis of multiple gene products from a single polycistronic mRNA using the Thermococcus kodakaraensis-based translation system

Previously, we have developed a system for cell-free protein synthesis operated at high temperatures using a lysate of thermococcus kodakaraensis. Here, we tested the synthesis of multiple gene products from a single mRNA. The selected target proteins were TK0944 and TK0943 proteins, the alpha-subunit and the szlig-subunit of acetyl-CoA synthetase III (ACS III) forming an intracellular enzyme complex composed of two beta- and two szlig-subunits (alpha
₂
szlig
₂
). To facilitate the detection and purification of synthesized protein, a His-tag was attached to the N-terminus of TK0944 protein (His-TK0944) and an HA-tag was attached to the C-terminus of TK0943 protein (TK0943-HA). By His-tag purification, only His-TK0944 protein was detected by SDS-PAGE analysis. On the other hand, TK0943-HA protein was detected in the fraction after cell-free protein synthesis. These results indicated that His-TK0944 and TK0943-HA proteins were simultaneously synthesized from a single polycistronic mRNA by using the T. kodakaraensis translation system.