To decrease the phosphorylation of eukaryotic translation factor eIF2α and thereby improving the capacity of cap-dependent translation initiation, the influence of the small component C38 (GSK2606414, GlaxoSmithKline, Dresden, Germany), a specific PERK inhibitor, was analyzed. Inhibitor containing lysate was preincubated for 10 min at RT before the cell-free reaction was started. To analyze the effect on the protein synthesis rate, the pIX4.0-Luc plasmid was added to the reaction and the yield of active luciferase was determined.
Synthesis in wheat germ lysate was performed in a transcription/translation coupled dialysis system using the RTS100 Wheat Germ CECF Kit (Biotechrabbit) for 24 h at 24 °C according to the manufacturer´s instructions. Again, 14C-leucine (2.47 dpm/pmol) was added to the reaction mixture to determine the size, yield and integrity of the de novo synthesized protein.
To obtain functional active hTERT enzyme, the assembly with a typical RNA component of telomerase is required [27 (link)]. The assembly was realized in two different ways: RNA was generated in a previous transcription reaction by using T7 RNA–polymerase followed by RNA purification using DyeEx spin columns (Qiagen). The freshly synthesized hTR RNA was directly added to the cell-free reaction (CHO and Sf21 cell-free reactions). Alternatively, a plasmid harboring the nucleotide sequence encoding the RNA component of human telomerase under control of the T7-promoter was added directly to the translation reaction (coupled reaction; wheat germ; final concentration of hTR plasmid: 20 ng/µL).