Editing Invitro Translation System (section)

=== Cell-free protein synthesis ===

Cell-free synthesis reactions were performed using CHO cell derived translationally active lysates. Lysates were prepared from cultured, exponentially grown CHO cells as described in previous studies&nbsp; [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042383/#pone.0163670.ref047 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042383/#pone.0163670.ref047]&nbsp; . The protein synthesis processes were conducted in coupled transcription/translation reactions in a final volume of 20 to 50 μl. The reactions were incubated in a thermomixer (Eppendorf) for 3 h at 30°C and 600 RPM. Protein synthesis reactions were composed of 40% (v/v) of translationally active lysate supplemented with HEPES-KOH (pH 7.5, f.c. 30 mM), sodium acetate (f.c. 100 mM), Mg(OAc)<sub>2</sub> (f.c. 3.9 mM), KOAc (f.c. 150 mM), amino acids (100 μM), spermidin (f.c. 0.25 mM), DTT (2.5 mM) and energy generation components including creatine phosphokinase (f.c. 0.1 mg/ml), creatine phosphate (20 mM), ATP (1.75 mM) and GTP (0.3 mM). If not reported otherwise, 60 ng/μl of plasmid DNA was added to the translation reaction. To enable DNA transcription to mRNA during cell-free protein synthesis, 1 U/μl T7 RNA polymerase, 0.3 mM of UTP and CTP and 0.1 mM of the cap analogue m7G(ppp)G were added to the reaction. Cell-free protein synthesis reactions were supplemented with radioactive <sup>14</sup>C leucine (f.c. 50 μM, specific radioactivity 66.67 dpm/pmol) for radiolabeling of produced proteins, in order to allow the further analysis of synthesized proteins by autoradiography and liquid scintillation counting.

Enrichment of membrane and secreted proteins into microsomal structures was investigated by repetitive translation cycles. After each translation step translation mixture was separated into microsomal fraction and supernatant by centrifugation (16000xg, 15 min, 4°C). A 5 μl aliquot of each fraction was taken for further analysis. Microsomal fraction was newly resuspended using freshly prepared translation mixture without vesicles to start a new translation cycle and thereby enabling multiple addressing of microsomes.
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