Individual eukaryotic cell-free protein synthesis systems differ in their composition and reaction conditions. Sf21- and CHO-based cell-free reactions have similar characteristics and were performed in a batch-formatted reaction mode. A 25 µL standard translation reaction of a Sf21 and CHO based cell-free synthesis was composed of 6 µL purified mRNA, 40% lysate, canonical amino acids (200 µM each), ATP (1.75 mM), GTP (0.45 mM) and 14C-labeled leucine (200 dpm/pmol) for the detection of de novo synthesized proteins. For the functional analysis of WNT proteins (β-catenin accumulation assay), proteins were synthesized in the absence of 14C-leucine. Protein translation reactions based on Sf21 lysates were incubated for 90 min at 27 °C, 600 rpm using a thermomixer (Eppendorf, Hamburg, Germany). Translation reactions based on CHO cell lysates were performed at 30 °C and 120 min with gentle shaking at 600 rpm. If required translation mixture (TM) of both cell-free reactions were further fractionated for analysis of protein translocation. The fractionation was realized by centrifugation at 16,000× g for 10 min at 4 °C in order to separate the ER-derived microsomal fraction (MF) of the cell lysate from the supernatant (S). The microsomal fraction was resuspended in PBS buffer without calcium and magnesium ions for further analysis.
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).
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).
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