Target protein synthesis was achieved using pIX2.0 plasmids in a linked system based on translationally active Sf21-lysates.
Transcription reactions were carried out similarly to the previously described tRNA transcription, using 60 µg/mL of purified plasmid DNA, NTPs and cap analogues. Generated mRNA was purified by gel filtration using either DyeEx2.0 spin columns (Qiagen, Hilden, Germany) or Nap5 columns (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions.
The standard batch translation reaction mixture, ranging in volumes from 5–500 µL, contained 40% (v/v) Sf21 lysate, approximately 400 µg/mL coding mRNA, canonical amino acids (200 µM), ATP (1.75 mM) and GTP (0.45 mM). For monitoring protein quality and quantity reaction mixtures were supplemented either with 14C-labeled leucine (310.0 mCi/mmol, PerkinElmer, Waltham MA, U.S.A.) or 14C-labeled mannose (55 mCi/mL, ARC Inc., St. Louis MO, U.S.A.). Translation reactions were operated at 27 °C and 500 rpm in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) for 90–120 minutes. As negative controls, standard translation reactions were performed without the addition of mRNA (no-template control, NTC).
Transcription reactions were carried out similarly to the previously described tRNA transcription, using 60 µg/mL of purified plasmid DNA, NTPs and cap analogues. Generated mRNA was purified by gel filtration using either DyeEx2.0 spin columns (Qiagen, Hilden, Germany) or Nap5 columns (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions.
The standard batch translation reaction mixture, ranging in volumes from 5–500 µL, contained 40% (v/v) Sf21 lysate, approximately 400 µg/mL coding mRNA, canonical amino acids (200 µM), ATP (1.75 mM) and GTP (0.45 mM). For monitoring protein quality and quantity reaction mixtures were supplemented either with 14C-labeled leucine (310.0 mCi/mmol, PerkinElmer, Waltham MA, U.S.A.) or 14C-labeled mannose (55 mCi/mL, ARC Inc., St. Louis MO, U.S.A.). Translation reactions were operated at 27 °C and 500 rpm in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) for 90–120 minutes. As negative controls, standard translation reactions were performed without the addition of mRNA (no-template control, NTC).