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Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

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Progress of selection for ligases at 65°C.The fraction of 32P-labelled cDNA that bound to streptavidin agarose after each round of selection is shown. The reaction time was either 60 min or 5 min as indicated by black or gray bars, respectively.
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pone-0112028-g002: Progress of selection for ligases at 65°C.The fraction of 32P-labelled cDNA that bound to streptavidin agarose after each round of selection is shown. The reaction time was either 60 min or 5 min as indicated by black or gray bars, respectively.

Mentions: To enrich for RNA ligase enzyme with increased thermostability, we performed a total of six rounds of selection and amplification (Figure 1A). After reverse transcription, the mRNA-displayed proteins were incubated with the HO-substrate-65 and the RNA splint for 60 min and/or 5 min. The percentage of cDNA that was immobilized on streptavidin beads after each round of selection is shown in Figure 2. In the case of the 60 minute incubation, the percentage of immobilized cDNA increased steadily over the course of the selection, from 0.61% after round 1 to 6.6% after round 6. In order to increase the selection pressure by favoring enzymes with faster ligation rates, in round 4, we incubated a second aliquot of the mRNA-displayed proteins for only 5 min yielding 0.66% immobilized cDNA. This cDNA was used as input for following round, but no increase in the amount of immobilized cDNA after 5 min incubation was observed in round 5 (amount decreased to 0.41%). Therefore, we performed the sixth and final round of selection, again with 60 min incubation. The resulting DNA was cloned and sequenced for further analysis.


Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Progress of selection for ligases at 65°C.The fraction of 32P-labelled cDNA that bound to streptavidin agarose after each round of selection is shown. The reaction time was either 60 min or 5 min as indicated by black or gray bars, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230948&req=5

pone-0112028-g002: Progress of selection for ligases at 65°C.The fraction of 32P-labelled cDNA that bound to streptavidin agarose after each round of selection is shown. The reaction time was either 60 min or 5 min as indicated by black or gray bars, respectively.
Mentions: To enrich for RNA ligase enzyme with increased thermostability, we performed a total of six rounds of selection and amplification (Figure 1A). After reverse transcription, the mRNA-displayed proteins were incubated with the HO-substrate-65 and the RNA splint for 60 min and/or 5 min. The percentage of cDNA that was immobilized on streptavidin beads after each round of selection is shown in Figure 2. In the case of the 60 minute incubation, the percentage of immobilized cDNA increased steadily over the course of the selection, from 0.61% after round 1 to 6.6% after round 6. In order to increase the selection pressure by favoring enzymes with faster ligation rates, in round 4, we incubated a second aliquot of the mRNA-displayed proteins for only 5 min yielding 0.66% immobilized cDNA. This cDNA was used as input for following round, but no increase in the amount of immobilized cDNA after 5 min incubation was observed in round 5 (amount decreased to 0.41%). Therefore, we performed the sixth and final round of selection, again with 60 min incubation. The resulting DNA was cloned and sequenced for further analysis.

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

Show MeSH