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Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins.

Iskakova MB, Szaflarski W, Dreyfus M, Remme J, Nierhaus KH - Nucleic Acids Res. (2006)

Bottom Line: The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20 degrees C.A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids.Accordingly, a second addition of amino acids after 10 h during an incubation at 20 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany.

ABSTRACT
Cell-free coupled transcription-translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 +/- 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20 degrees C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

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Total and active GFP synthesized at various conditions. Blue bars, total GFP synthesis; green bars, active GFP. (a) GFP synthesized after 20 h at 30°C of incubation in the presence of mutant T7 transcriptases and our reaction-mix preparation (semi-continuous system; 1 ml reaction volume; see Materials and Methods; the unusually low active fraction of almost 20% might be due to our preparation procedure of the S30 lysate). (b) GFP synthesis after 12 h in the RTS 100 (batch system; 10 μl reaction volume, wild-type T7 polymerase) at various incubation temperatures.
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fig2: Total and active GFP synthesized at various conditions. Blue bars, total GFP synthesis; green bars, active GFP. (a) GFP synthesized after 20 h at 30°C of incubation in the presence of mutant T7 transcriptases and our reaction-mix preparation (semi-continuous system; 1 ml reaction volume; see Materials and Methods; the unusually low active fraction of almost 20% might be due to our preparation procedure of the S30 lysate). (b) GFP synthesis after 12 h in the RTS 100 (batch system; 10 μl reaction volume, wild-type T7 polymerase) at various incubation temperatures.

Mentions: The SDS–PAGE analysis of the GFP production from the transcripts produced by either the double-mutants or by WT T7 RNAPs revealed that the yield of GFP dramatically decreased using the double-mutant variants and is almost undetectable in the case of the P266L/I810S mutant. On the contrary, the native-PAGE analysis of active GFP produced from these transcripts allows an easy detection of the GFP band in all cases, including the P266L/I810S double-mutant (summarized in Figure 2a). In spite of the low yield we can conclude that a reduction in the rate of transcription significantly improves the active fraction of the synthesized protein probably by re-establishing the coupling of transcription and translation.


Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins.

Iskakova MB, Szaflarski W, Dreyfus M, Remme J, Nierhaus KH - Nucleic Acids Res. (2006)

Total and active GFP synthesized at various conditions. Blue bars, total GFP synthesis; green bars, active GFP. (a) GFP synthesized after 20 h at 30°C of incubation in the presence of mutant T7 transcriptases and our reaction-mix preparation (semi-continuous system; 1 ml reaction volume; see Materials and Methods; the unusually low active fraction of almost 20% might be due to our preparation procedure of the S30 lysate). (b) GFP synthesis after 12 h in the RTS 100 (batch system; 10 μl reaction volume, wild-type T7 polymerase) at various incubation temperatures.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636455&req=5

fig2: Total and active GFP synthesized at various conditions. Blue bars, total GFP synthesis; green bars, active GFP. (a) GFP synthesized after 20 h at 30°C of incubation in the presence of mutant T7 transcriptases and our reaction-mix preparation (semi-continuous system; 1 ml reaction volume; see Materials and Methods; the unusually low active fraction of almost 20% might be due to our preparation procedure of the S30 lysate). (b) GFP synthesis after 12 h in the RTS 100 (batch system; 10 μl reaction volume, wild-type T7 polymerase) at various incubation temperatures.
Mentions: The SDS–PAGE analysis of the GFP production from the transcripts produced by either the double-mutants or by WT T7 RNAPs revealed that the yield of GFP dramatically decreased using the double-mutant variants and is almost undetectable in the case of the P266L/I810S mutant. On the contrary, the native-PAGE analysis of active GFP produced from these transcripts allows an easy detection of the GFP band in all cases, including the P266L/I810S double-mutant (summarized in Figure 2a). In spite of the low yield we can conclude that a reduction in the rate of transcription significantly improves the active fraction of the synthesized protein probably by re-establishing the coupling of transcription and translation.

Bottom Line: The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20 degrees C.A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids.Accordingly, a second addition of amino acids after 10 h during an incubation at 20 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany.

ABSTRACT
Cell-free coupled transcription-translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 +/- 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20 degrees C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

Show MeSH
Related in: MedlinePlus