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We examined the inhibitory effects of adding various detergents, solvents, protease inhibitors and other additives on the level of protein synthesis in a rabbit reticulocyte lysate-based cell-free expression system. |
Introduction
We determined the effect of adding various concentrations of additives and potential contaminants to rabbit reticulocyte lysate-based in vitro transcription/translation systems. We chose reagents that are added to the TNT® reaction to optimize protein expression levels and common contaminants in the DNA template preparation. The T7 Control DNA (Cat.# L4821), which encodes the firefly luciferase gene, was translated in the presence of increasing concentrations of the following agents: methanol, ethanol, DMSO, formamide, Nikkol, Tween®-20, NP-40, Triton® X-100, oxidized glutathione, leupeptin, pepstatin, aprotinin and Pefabloc® SC. These agents included various alcohols, detergents and protease inhibitors. These experiments were performed with the Gold TNT® T7 Express 96 System, but this information should also be applicable to other rabbit reticulocyte-based transcription/translation systems.
Materials and Methods
The Gold TNT® T7 Express 96 System (Cat.# L5600) includes a 96-well plate with 20μl per well of predispensed rabbit reticulocyte lysate that is evaluated for optimal transcriptional/translational activity. Reactions were assembled using the T7 Control DNA as a template as described in the Gold TNT® T7/SP6 Express 96 Systems Technical Manual (#TM054; 1). Each reaction contained 200ng of T7 Control DNA and 0.5μl of 1mM methionine in a total volume of 4μl, as well as 1μl of test compound, per well. Each test compound was examined in triplicate, and the final concentration of each compound is listed in Figures 1–3. The plates were incubated for 90 minutes at 30°C. Following translation, 1μl of each reaction was added to 99μl of 1X PBS in a white 96-well luminometry plate, followed by addition of 100μl of reconstituted Bright-Glo™ Luciferase Assay Reagent (Cat.# E2610). The plates were incubated for 10 minutes at room temperature, and luminescence was measured with a EG&G Berthold plate-reading luminometer with a 2-second read time per well.
Results and Conclusions
The average luminescence of triplicate reactions was used to calculate the decrease in luciferase activity in the presence of each compound, as compared to a control TNT® reaction with no additive (set as 100%). In general, increasing concentrations of each additive caused increasing inhibition of luciferase luminescence. Methanol, ethanol, DMSO, formamide (Figure 1), Nikkol, Tween®-20, NP-40, Triton® X-100 (Figure 2), oxidized glutathione and Pefabloc® SC (Figure 3) inhibited protein synthesis at higher concentrations, while leupeptin, aprotinin and pepstatin did not have a noticeable effect even at the higher concentrations (Figure 3). Since each translation reaction was diluted 1:200 into the luciferase assay, the decrease in observed luciferase activity is most likely due to decreased luciferase synthesis and not a direct inhibition of luciferase activity. However, direct inhibition is still a possibility and might explain the total lack of luciferase activity observed in the reaction with 4% formamide. The additives that inhibited luciferase expression by 50% or more are summarized in Table 1.
Other potential inhibitors, such as cap analog and common PCR enhancers, were examined in another series of experiments (2). For more information on optimizing your TNT® reactions, see Optimize Your TNT® Reticulocyte Lysate Systems Reactions.
Reference
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Figure 1. Effects of methanol (MeOH), ethanol (EtOH), DMSO and formamide (form) on luciferase expression in the Gold TNT® T7 Express 96 System. |
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Figure 2. Effect of Nikkol, Tween®-20 (Tween), NP-40 and Triton® X-100 (Triton X) on luciferase expression in the Gold TNT® T7 Express 96 System. |
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Figure 3. Effect of oxidized glutathione (GSSG), leupeptin (Leu), pepstatin (Pep), aprotinin (Aprot) and Pefabloc® SC (Pefa) on luciferase expression in the Gold TNT® T7 Express 96 System. |
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