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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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Various mRNA constructs in the coupled transcription–translation system. (a) Upper panel, stability elements of rRNA genes. Schematic representation of an rrn operon and major processing steps of the 16S and 23S rRNA. The drawing is not to scale. Primary processing cleavages by RNase III (lanes 3–7), and secondary processing to produce the mature termini of 16S rRNA (lane 1, 5′ end; lane 2, 3′ end) and 23S rRNA (lane 8, 5′ end; lane 9, 3′ end). Solid lines indicate mature RNAs; modified (36). (a) Lower panel, map of a plasmid used for mRNA stability test; derivative of a vector that contained a fragment of the rrnB operon including intergenic spacers. GFP was incorporated into the 23S rRNA sequence at AvaI cleavage sites into a position between nt 250 and 2773 (E.coli numbering, starting from 5′ end of 23S rRNA). Restriction sites and rrnB operon elements are indicated. (b) Total GFP synthesis from different plasmid–DNA constructs (semi-continuous system; reaction volume 1 ml). Squares, GFP-mRNA flanked by the 23S rRNA stability elements (stable); triangles, same as squares but with destroyed stability elements (unstable); closed diamonds, standard expression vector for GFP synthesis (GFP). For further explanations see text.
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fig3: Various mRNA constructs in the coupled transcription–translation system. (a) Upper panel, stability elements of rRNA genes. Schematic representation of an rrn operon and major processing steps of the 16S and 23S rRNA. The drawing is not to scale. Primary processing cleavages by RNase III (lanes 3–7), and secondary processing to produce the mature termini of 16S rRNA (lane 1, 5′ end; lane 2, 3′ end) and 23S rRNA (lane 8, 5′ end; lane 9, 3′ end). Solid lines indicate mature RNAs; modified (36). (a) Lower panel, map of a plasmid used for mRNA stability test; derivative of a vector that contained a fragment of the rrnB operon including intergenic spacers. GFP was incorporated into the 23S rRNA sequence at AvaI cleavage sites into a position between nt 250 and 2773 (E.coli numbering, starting from 5′ end of 23S rRNA). Restriction sites and rrnB operon elements are indicated. (b) Total GFP synthesis from different plasmid–DNA constructs (semi-continuous system; reaction volume 1 ml). Squares, GFP-mRNA flanked by the 23S rRNA stability elements (stable); triangles, same as squares but with destroyed stability elements (unstable); closed diamonds, standard expression vector for GFP synthesis (GFP). For further explanations see text.

Mentions: To test whether an increased mRNA stability improves the protein yield, we exploited the enormous stability of the ribosomal precursor RNA due to the long complementary sequences flanking the mature rRNA [(31); upper panel in Figure 3a]. We constructed a GFP-mRNA that is flanked by the highly conserved sequences enclosing the 23S rRNA and forms a strong base-paired stem resulting in a pseudo-circularization of the mRNA similar to that of the precursor rRNA (‘stable’ GFP-mRNA, lower panel in Figure 3a). We expected a prolonged mRNA half-life, since the endo RNase E prefers substrates with unpaired 5′ ends and the exo PNPase and RNase II are specific for single-stranded RNAs (32). As a control we used the same mRNA except that the mutation in the 5′ flanking region disrupts the complementarity and thus pseudo-circularized mRNA fails to be produced (‘unstable’ GFP-mRNA). The latter (in contrast to the former) has been shown to be resistant against in vitro RNase III cleavage, which is specific for secondary structures, thus revealing that no secondary structure has been formed (33). With both constructs, however, we observed levels of GFP production that were two times less than from our usual construct for GFP expression (Figure 3b). Owing to the low yield of GFP synthesis we did not pursue further the question whether indeed the ‘stable’ construct provided a more stable mRNA.


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)

Various mRNA constructs in the coupled transcription–translation system. (a) Upper panel, stability elements of rRNA genes. Schematic representation of an rrn operon and major processing steps of the 16S and 23S rRNA. The drawing is not to scale. Primary processing cleavages by RNase III (lanes 3–7), and secondary processing to produce the mature termini of 16S rRNA (lane 1, 5′ end; lane 2, 3′ end) and 23S rRNA (lane 8, 5′ end; lane 9, 3′ end). Solid lines indicate mature RNAs; modified (36). (a) Lower panel, map of a plasmid used for mRNA stability test; derivative of a vector that contained a fragment of the rrnB operon including intergenic spacers. GFP was incorporated into the 23S rRNA sequence at AvaI cleavage sites into a position between nt 250 and 2773 (E.coli numbering, starting from 5′ end of 23S rRNA). Restriction sites and rrnB operon elements are indicated. (b) Total GFP synthesis from different plasmid–DNA constructs (semi-continuous system; reaction volume 1 ml). Squares, GFP-mRNA flanked by the 23S rRNA stability elements (stable); triangles, same as squares but with destroyed stability elements (unstable); closed diamonds, standard expression vector for GFP synthesis (GFP). For further explanations see text.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Various mRNA constructs in the coupled transcription–translation system. (a) Upper panel, stability elements of rRNA genes. Schematic representation of an rrn operon and major processing steps of the 16S and 23S rRNA. The drawing is not to scale. Primary processing cleavages by RNase III (lanes 3–7), and secondary processing to produce the mature termini of 16S rRNA (lane 1, 5′ end; lane 2, 3′ end) and 23S rRNA (lane 8, 5′ end; lane 9, 3′ end). Solid lines indicate mature RNAs; modified (36). (a) Lower panel, map of a plasmid used for mRNA stability test; derivative of a vector that contained a fragment of the rrnB operon including intergenic spacers. GFP was incorporated into the 23S rRNA sequence at AvaI cleavage sites into a position between nt 250 and 2773 (E.coli numbering, starting from 5′ end of 23S rRNA). Restriction sites and rrnB operon elements are indicated. (b) Total GFP synthesis from different plasmid–DNA constructs (semi-continuous system; reaction volume 1 ml). Squares, GFP-mRNA flanked by the 23S rRNA stability elements (stable); triangles, same as squares but with destroyed stability elements (unstable); closed diamonds, standard expression vector for GFP synthesis (GFP). For further explanations see text.
Mentions: To test whether an increased mRNA stability improves the protein yield, we exploited the enormous stability of the ribosomal precursor RNA due to the long complementary sequences flanking the mature rRNA [(31); upper panel in Figure 3a]. We constructed a GFP-mRNA that is flanked by the highly conserved sequences enclosing the 23S rRNA and forms a strong base-paired stem resulting in a pseudo-circularization of the mRNA similar to that of the precursor rRNA (‘stable’ GFP-mRNA, lower panel in Figure 3a). We expected a prolonged mRNA half-life, since the endo RNase E prefers substrates with unpaired 5′ ends and the exo PNPase and RNase II are specific for single-stranded RNAs (32). As a control we used the same mRNA except that the mutation in the 5′ flanking region disrupts the complementarity and thus pseudo-circularized mRNA fails to be produced (‘unstable’ GFP-mRNA). The latter (in contrast to the former) has been shown to be resistant against in vitro RNase III cleavage, which is specific for secondary structures, thus revealing that no secondary structure has been formed (33). With both constructs, however, we observed levels of GFP production that were two times less than from our usual construct for GFP expression (Figure 3b). Owing to the low yield of GFP synthesis we did not pursue further the question whether indeed the ‘stable’ construct provided a more stable mRNA.

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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