Invitro Translation System

Preparation of Cell Lysate

https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.25013 CHO cells were grown exponentially in suspension culturesin well-controlled fermenters at 37�C using the Power CHO-2 chemically defined, serum-free medium (Lonza, Cologne,Germany). CHO cells were harvested at a density ofapproximately 2�106cells/mL. Collected cells were centri-fuged at 200gfor 10 min and washed once with a bufferconsisting of 40 mM HEPES-KOH (pH 7.5), 100 mMNaOAc, and 4 mM DTT. The cell pellet was resuspendedto achieve a density of approximately 5�108cells/mL. Celldisruption was accomplished by syringing the harvested cellpellet through a 20-gauge needle, followed by centrifugationat 10,000gfor 10 min to remove the nuclei and cell debris.The supernatant was applied to a size-exclusion chromatography column (Sephadex G-25, GE Healthcare,Freiburg, Germany) and the elution fractions (each 1 ml)with an RNA content above an absorbance of 100 at 260 nmwere pooled. Residual mRNA was digested by micrococcalnuclease (S7) treatment. In this respect, 10 U/mL S7 nuclease(Roche, Mannheim, Germany) and 1 mM CaCl2(finalconcentration) were added to the eluate and the reactionmixture was incubated for 2 min at room temperature. Thereaction was inactivated by the addition of 6.7 mM EGTA(f.c.). Finally, translationally active lysates were immediatelyshock-frozen in liquid nitrogen and stored at�80�C in orderto preserve maximum activity.</p>

Cell-Free Protein Synthesis

Coupled transcription–translation reactions were performedin 25mL batch volume. Protein production was operated at33�C in a thermomixer (Thermomixer comfort, Eppendorf,Hamburg, Germany) with gentle shaking at 500 rpm.Reactions were composed of 25% (v/v) CHO cell lysate,canonical amino acids (100mM), nucleoside triphosphates(1.75 mM ATP, 0.30 mM CTP, 0.30 mM GTP, and 0.30 mMUTP), vector DNA and 1 U/mLT7 RNA-polymerase (Agilent,Waldbronn, Germany). To monitor protein quality andquantity, reaction mixtures were supplemented with14C-labeled leucine (specific radioactivity 75.0 dpm/pmol). As acomparison of the productivity, commercially availablecoupled cell-free expression systems based onSpodopterafrugiperda21 cells (TNT T7 Insect Cell Extract ProteinExpression System, Promega, Mannheim, Germany), rabbitreticulocytes (TNT T7 Coupled Reticulocyte Lysate System,Promega), and wheat germ (TNT T7 Coupled Wheat GermExtract System, Promega) were purchased and in vitroreactions were performed according to the manufacturer’sinstructions. All systems were used with the recommendedindividual LUC-encoding expression template provided by themanufacturer. In the case of the TNT T7 Coupled ReticulocyteLysate System, the provided Luciferase T7 Control DNA wasapplied. The TNT T7 Coupled Wheat Germ Extract Systemand the TNT T7 Insect Cell Extract Protein Expression Systemwere used with the provided T7 Linear Control DNA andLuciferase ICE T7 Control DNA, respectively.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163670

Template generation

Cell-free protein syntheses in CHO lysates were performed using either vector DNA or linear DNA templates generated by polymerase chain reaction (PCR). Different vector backbones harboring a firefly luciferase (Luc) gene and internal ribosomal entry sites were tested in cell-free systems. This study included the following plasmids: pIX3.0-CRPV(GCT)-Luc, pIX3.0-EMCV-Luc, pcDNA3.1-CRPV(GCT)-Luc and pT7CEF1-EMCV-Luc. If not stated differently all vector based DNA constructs are based on a common in vitro protein synthesis pIX3.0 vector backbone (Biotech Rabbit) containing regulatory sequences for transcription and translation. A T7 promotor and a T7 terminator constituted regulatory sequences for transcription. For translation initiation an internal ribosomal entry site (IRES) of cricket paralysis virus was included into the 5´UTR and the start codon triplet of the gene of interest was exchanged from ATG to GCT, as it was shown in previous studies (Broedel et. al. 2014). The following proteins were selected as model proteins for initial tests: Luciferase (Luc) (cytosolic protein, 60.6 kDa), enhanced yellow fluorescent protein eYFP (cytosolic protein, 26.9 kDa) and erythropoietin including melittin signal peptide (Mel-EPO) for efficient translocation of proteins into microsomal structures of the CHO lysates (glycosylated protein, 19.7 kDa) with two deleted N-glycosylation sites at asparagine 52 and glutamine 110. To evaluate the performance of the CHO lysate cell-free protein synthesis system, different classes of membrane proteins were synthesized covering ion channels, G protein coupled receptors as well as multi-pass transmembrane proteins. During initial experiments different vector backbones and IRES were evaluated regarding their performance in CHO lysate based cell-free systems.

Templates for analyzing the ability of the CHO based cell-free system to synthesize proteins from linear DNA constructs were amplified by PCR. PCR was performed by using Hot Star HiFidelity polymerase (Qiagen) and following the manufacturer´s protocol. For this purpose the plasmids pIX3.0-CRPV(GCT)-Luc and pIX3.0-CRPV(GCT)-EPO were used in the PCR as general DNA templates. Adapter primers N-0 (5´ATG ATA TCT CGA GCG GCC GCT AGC TAA TAC GAC TCA CTA TAG GGA GAC CAC AAC GGT TTC CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATA AAC AAT G 3´) and C-0 (TAA TAA CTA ACT AAC CAA GAT CTG TAC CCC TTG GGG CCT CTA AAC GGG TCT TGA GGG GTT TTT TGG ATC CGA ATT CAC CGG TGA TAT CAT) were used to amplify linear DNA-templates. Each of the adapter primers has overlap sequences which fuse regulatory sequences necessary for translation and transcription to the gene of interest.

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  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042383/#pone.0163670.ref047  . 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)₂ (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 ¹⁴C 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.