(A) Detection of the synthesis of target proteins (indicated at the bottom of lane 2–7) with [³⁵S] methionine labeling in the reconstituted T. thermophilus system. As a control, mRNA is not added (lane 1, mRNA). BstYI (23 kDa) and PspGI (32 kDa): restriction enzymes from Bacillus stearothermophilus and Pyroccocus species, respectively; TAP (57 kDa): an alkaline phosphatase from T. thermophilus; AMase: (59 kDa): an amylomaltase from T. thermophilus; Vent DNAP (90 kDa): a DNA polymerase from hyperthermophilc Thermocuccus litoralis; stGFP (29 kDa): an engineered thermostable GFP variant from this study. The MW standards (MW, kDa) are indicated on the left. (B) Synthesis of Vent DNAP with [³⁵S] methionine labeling at different temperatures in the reconstituted T. thermophilus system. (C) Comparison of the stability of mRNA at 60°C in in vitro translation reactions of the reconstituted T. thermophilus system (left panel) or T. thermophilus cell extract (right panel). The stability of ³²P-labeled BstYI mRNA at various time points (indicated on top) is analyzed on a SDS-PAGE gel, followed by autoradiography. Purified ³²P-labeled BstYI mRNA without incubation is shown as the control (ctrl). The RNA standards in base pairs (bp) are indicated on the left.