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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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In vitro selection of artificial ligase enzymes with increased stability.(A) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C [17], [22]. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated in vitro yielding a library of mRNA-displayed proteins [22]. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (B) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C [17], [22], and with (C) 20 nucleotides of each substrate during the current selection at 65°C. HEG4 represents the linker of four hexaethylene glycol units (red wavy line).
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pone-0112028-g001: In vitro selection of artificial ligase enzymes with increased stability.(A) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C [17], [22]. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated in vitro yielding a library of mRNA-displayed proteins [22]. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (B) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C [17], [22], and with (C) 20 nucleotides of each substrate during the current selection at 65°C. HEG4 represents the linker of four hexaethylene glycol units (red wavy line).

Mentions: Sequence analysis of the artificial RNA ligase enzymes that resulted from the final round of the previous in vitro selection performed at 23°C [17] revealed substantial sequence diversity. The DNA encoding those diverse ligases was used as the starting library for the selection at 65°C described in this paper without introducing further sequence diversity. The RNA ligation reaction catalyzed by the previously selected enzymes was dependent on a complementary splint oligonucleotide that base-pairs to the two substrate RNAs [17] (Figure 1). During the selection at 23°C, this splint base-paired to eight nucleotides of each substrate (Figure 1B). In order to ensure stable base-pairing during a splinted ligation at elevated temperatures, a longer splint was chosen to extend the region complementary to each substrate to twenty nucleotides (Figure 1C). The 40-nucleotide-long splint resulted in a melting temperature of 76°C and 69°C with the PPP-substrate and the HO-substrate, respectively (Figure S1).


Thermostable artificial enzyme isolated by in vitro selection.

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

In vitro selection of artificial ligase enzymes with increased stability.(A) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C [17], [22]. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated in vitro yielding a library of mRNA-displayed proteins [22]. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (B) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C [17], [22], and with (C) 20 nucleotides of each substrate during the current selection at 65°C. HEG4 represents the linker of four hexaethylene glycol units (red wavy line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230948&req=5

pone-0112028-g001: In vitro selection of artificial ligase enzymes with increased stability.(A) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C [17], [22]. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated in vitro yielding a library of mRNA-displayed proteins [22]. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (B) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C [17], [22], and with (C) 20 nucleotides of each substrate during the current selection at 65°C. HEG4 represents the linker of four hexaethylene glycol units (red wavy line).
Mentions: Sequence analysis of the artificial RNA ligase enzymes that resulted from the final round of the previous in vitro selection performed at 23°C [17] revealed substantial sequence diversity. The DNA encoding those diverse ligases was used as the starting library for the selection at 65°C described in this paper without introducing further sequence diversity. The RNA ligation reaction catalyzed by the previously selected enzymes was dependent on a complementary splint oligonucleotide that base-pairs to the two substrate RNAs [17] (Figure 1). During the selection at 23°C, this splint base-paired to eight nucleotides of each substrate (Figure 1B). In order to ensure stable base-pairing during a splinted ligation at elevated temperatures, a longer splint was chosen to extend the region complementary to each substrate to twenty nucleotides (Figure 1C). The 40-nucleotide-long splint resulted in a melting temperature of 76°C and 69°C with the PPP-substrate and the HO-substrate, respectively (Figure S1).

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
Related in: MedlinePlus