Cell-free synthesis of GFP under high temperature conditions

Previously, we have developed a system for cell-free protein synthesis that can be operated at high temperatures using a lysate of Thermococcus kodakaraensis. Here, we tested cell-free synthesis of green fluorescent protein (GFP) using the T. kodakaraensis system. A thermostable GFP derivative (tGFP) was used as a reporter protein. By changing the codon usage of tGFP gene for T. kodakaraensis, production of tGFP was detectable in a temperature range of 50degC to 65degC, with an optimum at 60degC. In this condition, active tGFP constitute only 34-62 % of the total protein synthesized. The ratio of active tGFP synthesized markedly increased to 77-84 % by the addition of T. kodakaraensis chaperonin (CpkB) oligomers at 60degC. As tGFP, once folded properly, showed a high stability under these conditions, the results here clearly indicate the presence of a heat-labile state(s) in the folding process of tGFP.