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Focus: Inhibitors of TNT® Cell-Free Protein Expression Systems
Effects of Various Additives or Contaminants on in vitro Transcription/Translation in
the Gold TNT® T7 Express 96 System
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. |
By Natalie Betz, Ph.D.
Promega Corporation
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.
Table 1. Concentration of
Each Additive that Inhibits Luciferase Expression by ≥50%. |
Additive |
Concentration to
Inhibit Luciferase Synthesis by ≥50% |
| DMSO |
4%
|
| Formamide
|
2%
|
| Oxidized glutathione |
5mM
|
| Nikkol
|
0.05%
|
| Ethanol
|
4%
|
|
Tween®-20
|
0.5%
|
| Pefabloc®
SC
|
1mg/ml
|
| NP-40
|
0.05%
|
| Triton® X-100
|
0.1%
|
| Methanol
|
4%
|
Reference
- Gold TNT® T7/SP6 Express 96 Systems Technical
Manual (#TM054), Promega Corporation.
- Betz, N. (2001) Characterization of TNT® T7
Quick for PCR DNA. Promega Notes 77, 19–22.
Products may be covered by pending or issued patents or may have certain limitations
on use. Please visit our patent and trademark web page for more information.
TNT is a trademark of Promega Corporation and is registered with the U.S. Patent and
Trademark Office. Bright-Glo is a trademark of Promega Corporation.
Pefabloc is a registered trademark of Pentapharm Ltd. Corporation. Tween is a registered trademark of ICI
Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals & Plastics Technology
Corporation.
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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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