Optimized Cell-Free Protein Synthesis

Sf21 lysate preparation and coupled CFPS have already been described and explained (Brödel et al., 2013b (link); Quast et al., 2015 (link)). Minor changes were made during lysate preparation. In all buffers used during the lysate preparation, DTT was omitted, and 0.05 mM GSH and 0.25 mM GSSG were added. Coupled transcription-translation reactions were performed in a batch mode format. Protein synthesis was performed in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 21°C (if not stated otherwise) and with gentle shaking at 500 rpm for 3 h. Reaction volumes of 50 μL were composed of 40% (v/v) Sf21 cell lysate, 100 µM of each canonical amino acid, nucleoside triphosphates (1.75 mM ATP, 0.30 mM CTP, 0.30 mM GTP, and 0.30 mM UTP), 100 ng/μL vector DNA, and 1 U/µL T7 RNA-polymerase (Agilent, Waldbronn, Germany), 0–75 µM porcine hemin (Alfa Aesar, Haverhill, USA) dissolved in NaOH. To monitor protein quality and quantity, reaction mixtures were supplemented with ¹⁴C-labeled leucine (Perkin Elmer, Inc.; Baesweiler, Germany). No template controls (NTC) were prepared in the same way as the samples except that the DNA template was replaced by water.