Residue specific fluorescence labeling of Mel-EPO and KCSA in cell-free systems based on translationally active CHO cell lysate.

<p>Cell-free protein synthesis based on CHO cell extracts was carried out in the presence of BODIPY-TMR-tRNA(Phe) to allow the fluorescence labeling of <i>de novo</i> synthesized Mel-EPO and KCSA. Produced glycosylated and transmembrane proteins were separated by SDS-PAGE. In-gel fluorescence was detected using a variable mode imager (Typhoon Trio Plus, GE Healthcare).</p

Protein yields using different luciferase encoding plasmids in cell-free synthesis based on CHO cell lysates.

<p>Protein yields using different luciferase encoding plasmids in cell-free synthesis based on CHO cell lysates.</p

Analysis of various types of proteins synthesized in CHO cell lysates.

<p>Standard cell-free protein synthesis was performed using pIX3.0-CPRV(GCT) plasmid backbones containing the different genes of interest. ¹⁴C leucine labeled proteins were precipitated in acetone, separated by SDS-PAGE and visualized by autoradiography. Produced proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163670#pone.0163670.t002" target="_blank">Table 2</a>. Arrows indicate the expected protein band of each individual protein. No template control (NTC) contains translation mixture without template to visualize the background translational activity of the CHO lysate.</p

Enrichment of membrane proteins in microsomal fractions of CHO lysate using a repetitive vesicle addressing procedure.

<p>During the first synthesis step (Cycle 1) Mel-EPO and OPMR1 protein was produced according to the optimized conditions (3 U/μl T7 RNA polymerase). After completing the cell-free reaction, translation mixture was separated into microsomal fraction and supernatant by centrifugation (15 min, 4°C and 16000xg) in a standard table top centrifuge. Supernatant was removed and microsomal fraction was resuspended using freshly prepared translation mixture containing CHO cell lysate without microsomal structures to initiate the second cell-free translation cycle (Cycle 2). This procedure was repeated again, after finishing the second translation step to obtain a third step of addressing the microsomal fraction (Cycle 3). Samples of translation mixture (TM), supernatant (S) and microsomal fraction (MF) were collected after each cycle for further analysis. Protein yields were quantified by hot TCA precipitation of ¹⁴C leucine labeled cell-free produced proteins followed by scintillation measurement. Error bars show standard deviations calculated from triplicate analysis. Molecular weight and modifications of proteins were visualized by SDS-PAGE separation and autoradiography.</p

Time course of ¹⁴C-leucine labeled eYFP synthesized in batch and CECF mode.

<p>Cell-free reactions using insect lysate were carried out in the presence (+) and absence (−) of insect vesicles (V) and caspase inhibitor (CI). Translation mixtures were analyzed by SDS-PAGE and autoradiography. Cell-free synthesized eYFP shows a migration pattern corresponding to its expected molecular mass (calculated molecular mass  = 29 kDa).</p

Influence of PEG on protein production in cell-free systems based on translationally active CHO lysate.

<p>Two concentrations (1%, 2%) of different PEG molecules (3350, 5000, 20000) were analyzed in cell-free protein synthesis reactions using pIX3.0-CRPV(GCT)-eYFP plasmid template. Translation reactions without the addition of PEG but with supplementation of 3 U/μl (increased concentration) and 1 U/μl (standard concentration) T7 RNA polymerase served as control reactions <b><i>A</i></b>. Fluorescence signals of synthesized eYFP proteins were detected by fluorescence imaging on μ-Ibidi slides using the Typhoon Trio Plus Imager. <b><i>B</i></b>. Quantification of fluorescence signals was accomplished by using Image Quant TL Array analysis software. Error bars show standard deviations calculated from triplicate analysis.</p

Linear DNA templates in cell-free protein synthesis based on CHO cell lysate.

<p>Linear IRES-luciferase and IRES-Mel-EPO templates tested during cell-free protein synthesis reactions in the presence of ¹⁴C leucine. Different concentrations of linear DNA product and T7 RNA polymerase (Pol) were added to the individual reactions. Protein yield was quantified by hot TCA precipitation followed by scintillation measurement. Error bars show standard deviations calculated from triplicates.</p

Quantitative analysis of cell-free protein synthesis: A comparison of different luciferase encoding vector backbones probed in CHO cell lysates.

<p><b>A.</b> Total protein yields were determined by incorporation of radioactive ¹⁴C leucine into <i>de novo</i> synthesized proteins followed by hot TCA precipitation and scintillation measurement. Yields of active luciferase were quantified by standard luciferase assay. <b>B.</b> Western blot analysis of cell-free produced luciferase using primary Anti-Luc antibody (concentration 1:1000) and secondary Anti-rabbit-HRP conjugate antibody (1:2000). Analysis of luciferase bands was performed by using ECL reagent and detection of corresponding light emission. Error bars show standard deviations calculated from triplicate analysis. NTC sample contains translation mixture without synthesized protein.</p

“Difficult-to-express” proteins synthesized in the CHO lysate based cell-free system.

<p>“Difficult-to-express” proteins synthesized in the CHO lysate based cell-free system.</p

Fluorescence analysis of eYFP-tagged proteins synthesized in coupled <i>Sf</i>21, CHO and K562 cell-free expression systems.

<p>The image depicts the <i>de novo</i> synthesized secreted protein Mel-eYFP as well as the membrane proteins Mel-Hb-EGF-eYFP and Mel-EGFR-eYFP. Plasmids encoding the target proteins were equipped with (+) or without (−) the CrPV IGR IRES (GCU). Numbers indicate the increase of the expression levels using the CrPV IGR IRES-based construct compared to the no IRES control. No template controls were prepared in the same way as the samples, but instead of a DNA template, RNase-free water was added to the reaction. Samples were analyzed using the phosphorimager system (Typhoon TRIO+ Imager, GE Healthcare).</p