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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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Sequence alignment of the library used as input for the original ligase selection with ligases #6, #7 [17] and 10C that were selected at 23°C and at 65°C, respectively [41], [42].The amino acids in regions 1 and 2 of the original library (on top) were randomized prior to the selection at 23°C and are shown as “x” [43]. Dashes symbolize amino acids that are identical to the starting library. A period highlighted in gray represents a deletion. The underlined N-terminal amino acids of the library and ligase 10C represent a Flag epitope tag and an E epitope tag, respectively.
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pone-0112028-g003: Sequence alignment of the library used as input for the original ligase selection with ligases #6, #7 [17] and 10C that were selected at 23°C and at 65°C, respectively [41], [42].The amino acids in regions 1 and 2 of the original library (on top) were randomized prior to the selection at 23°C and are shown as “x” [43]. Dashes symbolize amino acids that are identical to the starting library. A period highlighted in gray represents a deletion. The underlined N-terminal amino acids of the library and ligase 10C represent a Flag epitope tag and an E epitope tag, respectively.

Mentions: The sequence of ligase 10C shared similarities to ligases #6 and #7 from the original selection with #7 being more similar (Figure 3). All three ligases were almost identical in sequence in the formerly randomized region 2, and all three shared the deletion of 17 amino acids following region 1. Ligases 10C and #7 also shared the sequence in region 1, but 10C contained a second deletion of 13 amino acids near the C-terminus. This C-terminal deletion was also found in other clones from the selection at 23°C [17], but these proteins were poorly soluble when expressed without an maltose-binding protein fusion and therefore unsuited for a direct comparison.


Thermostable artificial enzyme isolated by in vitro selection.

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

Sequence alignment of the library used as input for the original ligase selection with ligases #6, #7 [17] and 10C that were selected at 23°C and at 65°C, respectively [41], [42].The amino acids in regions 1 and 2 of the original library (on top) were randomized prior to the selection at 23°C and are shown as “x” [43]. Dashes symbolize amino acids that are identical to the starting library. A period highlighted in gray represents a deletion. The underlined N-terminal amino acids of the library and ligase 10C represent a Flag epitope tag and an E epitope tag, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112028-g003: Sequence alignment of the library used as input for the original ligase selection with ligases #6, #7 [17] and 10C that were selected at 23°C and at 65°C, respectively [41], [42].The amino acids in regions 1 and 2 of the original library (on top) were randomized prior to the selection at 23°C and are shown as “x” [43]. Dashes symbolize amino acids that are identical to the starting library. A period highlighted in gray represents a deletion. The underlined N-terminal amino acids of the library and ligase 10C represent a Flag epitope tag and an E epitope tag, respectively.
Mentions: The sequence of ligase 10C shared similarities to ligases #6 and #7 from the original selection with #7 being more similar (Figure 3). All three ligases were almost identical in sequence in the formerly randomized region 2, and all three shared the deletion of 17 amino acids following region 1. Ligases 10C and #7 also shared the sequence in region 1, but 10C contained a second deletion of 13 amino acids near the C-terminus. This C-terminal deletion was also found in other clones from the selection at 23°C [17], but these proteins were poorly soluble when expressed without an maltose-binding protein fusion and therefore unsuited for a direct comparison.

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