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David S. Waugh - One of the best experts on this subject based on the ideXlab platform.
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Differential temperature dependence of Tobacco Etch Virus and rhinoVirus 3C proteases
Analytical biochemistry, 2013Co-Authors: Sreejith Raran-kurussi, József Tözsér, Scott Cherry, Joseph E. Tropea, David S. WaughAbstract:Because of their stringent sequence specificity, the 3C-like proteases from Tobacco Etch Virus (TEV) and human rhinoVirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4 C, the temperature at which fusion protein substrates are usually digested. Here we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinoVirus 3C protease is approximately the same at its optimum temperature (30 C) and at 4 C, TEV protease is 10-fold less active at the latter temperature due primarily to a reduction in kcat.
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Tobacco Etch Virus proteinase: crystal structure of the active enzyme and its inactive mutant
Bioorganicheskaia khimiia, 2003Co-Authors: J. Phan, Rachel B. Kapust, Joseph E. Tropea, Artem G. Evdokimov, Alexander Wlodawer, David S. WaughAbstract:Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S 3–S 6. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.
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Structural basis for the substrate specificity of Tobacco Etch Virus protease.
The Journal of biological chemistry, 2002Co-Authors: Jason Phan, Rachel B. Kapust, Joseph E. Tropea, Alexander Zdanov, Artem G. Evdokimov, Howard K. Peters, Alexander Wlodawer, David S. WaughAbstract:Because of its stringent sequence specificity, the 3C-type protease from Tobacco Etch Virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.
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The P1' specificity of Tobacco Etch Virus protease.
Biochemical and biophysical research communications, 2002Co-Authors: Rachel B. Kapust, József Tözsér, Terry D. Copeland, David S. WaughAbstract:Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the Tobacco Etch Virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
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Tobacco Etch Virus protease mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency
Protein Engineering, 2001Co-Authors: Rachel B. Kapust, József Tözsér, Scott Cherry, Terry D. Copeland, Eric D Anderson, David S. WaughAbstract:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from Tobacco Etch Virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1 positions of the internal cleavage site and assessed their impact on the enzyme’s stability and catalytic activity. One of the P1 mutants, S219V, was not only far more stable than the wild-type protease (∼100-fold), but also a more efficient catalyst.
S Laín - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic activity of plum pox Virus-Tobacco Etch Virus chimeric NIa proteases.
FEBS letters, 1991Co-Authors: J A García, S LaínAbstract:Plasmids encoding chimeric NIa-type proteases made of sequences from the potyViruses plum pox Virus (PPV) and Tobacco Etch Virus (TEV) have been constructed. Their proteolytic activity on the large nuclear inclusion protein (NIb)-capsid protein (CP) junction of each Virus was assayed in Escherichia coli cells. The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition. In spite of the large homology among the PPV and TEV NIa-type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity. Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid. The results suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end. This region includes the conserved His at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gln at position -1 of the cleavage sites.
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Proteolytic activity of plum pox Virus—Tobacco Etch Virus chimeric NIa proteases
FEBS Letters, 1991Co-Authors: J A García, S LaínAbstract:Plasmids encoding chimeric NIa-type proteases made of sequences from the polyViruses plum pox Virus (PPV) and Tobacco Etch Virus (TEV) have been constructed. Their proteolytic activity on the large nuclear inclusion protein (NIa)-capsid protein (CP) junction of each Virus was assayed in Escherichia coli cells. The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition. In spite of the large homology among the PPV and TEV NIa-type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity. Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid. The results suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end. This region includes the conserved His at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gin at position −1 of the cleavage sites.
Rachel B. Kapust - One of the best experts on this subject based on the ideXlab platform.
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Tobacco Etch Virus proteinase: crystal structure of the active enzyme and its inactive mutant
Bioorganicheskaia khimiia, 2003Co-Authors: J. Phan, Rachel B. Kapust, Joseph E. Tropea, Artem G. Evdokimov, Alexander Wlodawer, David S. WaughAbstract:Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S 3–S 6. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.
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Structural basis for the substrate specificity of Tobacco Etch Virus protease.
The Journal of biological chemistry, 2002Co-Authors: Jason Phan, Rachel B. Kapust, Joseph E. Tropea, Alexander Zdanov, Artem G. Evdokimov, Howard K. Peters, Alexander Wlodawer, David S. WaughAbstract:Because of its stringent sequence specificity, the 3C-type protease from Tobacco Etch Virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.
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The P1' specificity of Tobacco Etch Virus protease.
Biochemical and biophysical research communications, 2002Co-Authors: Rachel B. Kapust, József Tözsér, Terry D. Copeland, David S. WaughAbstract:Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the Tobacco Etch Virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
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Tobacco Etch Virus protease mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency
Protein Engineering, 2001Co-Authors: Rachel B. Kapust, József Tözsér, Scott Cherry, Terry D. Copeland, Eric D Anderson, David S. WaughAbstract:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from Tobacco Etch Virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1 positions of the internal cleavage site and assessed their impact on the enzyme’s stability and catalytic activity. One of the P1 mutants, S219V, was not only far more stable than the wild-type protease (∼100-fold), but also a more efficient catalyst.
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Tobacco Etch Virus protease mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency
Protein Engineering, 2001Co-Authors: Rachel B. Kapust, József Tözsér, Scott Cherry, Terry D. Copeland, Eric D Anderson, Jeffrey D Fox, David S. WaughAbstract:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from Tobacco Etch Virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.
Daniel R. Gallie - One of the best experts on this subject based on the ideXlab platform.
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cap independent translation of Tobacco Etch Virus is conferred by an rna pseudoknot in the 5 leader
Journal of Biological Chemistry, 2005Co-Authors: Vladimir Zeenko, Daniel R. GallieAbstract:The Tobacco Etch Virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.
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Cap-independent Translation of Tobacco Etch Virus Is Conferred by an RNA Pseudoknot in the 5′-Leader
The Journal of biological chemistry, 2005Co-Authors: Vladimir Zeenko, Daniel R. GallieAbstract:The Tobacco Etch Virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.
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Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent
Journal of virology, 2001Co-Authors: Daniel R. GallieAbstract:The 5′ leader of Tobacco Etch Virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.
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cap independent translation conferred by the 5 leader of Tobacco Etch Virus is eukaryotic initiation factor 4g dependent
Journal of Virology, 2001Co-Authors: Daniel R. GallieAbstract:The 5′ leader of Tobacco Etch Virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.
József Tözsér - One of the best experts on this subject based on the ideXlab platform.
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Differential temperature dependence of Tobacco Etch Virus and rhinoVirus 3C proteases
Analytical biochemistry, 2013Co-Authors: Sreejith Raran-kurussi, József Tözsér, Scott Cherry, Joseph E. Tropea, David S. WaughAbstract:Because of their stringent sequence specificity, the 3C-like proteases from Tobacco Etch Virus (TEV) and human rhinoVirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4 C, the temperature at which fusion protein substrates are usually digested. Here we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinoVirus 3C protease is approximately the same at its optimum temperature (30 C) and at 4 C, TEV protease is 10-fold less active at the latter temperature due primarily to a reduction in kcat.
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The P1' specificity of Tobacco Etch Virus protease.
Biochemical and biophysical research communications, 2002Co-Authors: Rachel B. Kapust, József Tözsér, Terry D. Copeland, David S. WaughAbstract:Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the Tobacco Etch Virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
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Tobacco Etch Virus protease mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency
Protein Engineering, 2001Co-Authors: Rachel B. Kapust, József Tözsér, Scott Cherry, Terry D. Copeland, Eric D Anderson, David S. WaughAbstract:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from Tobacco Etch Virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1 positions of the internal cleavage site and assessed their impact on the enzyme’s stability and catalytic activity. One of the P1 mutants, S219V, was not only far more stable than the wild-type protease (∼100-fold), but also a more efficient catalyst.
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Tobacco Etch Virus protease mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency
Protein Engineering, 2001Co-Authors: Rachel B. Kapust, József Tözsér, Scott Cherry, Terry D. Copeland, Eric D Anderson, Jeffrey D Fox, David S. WaughAbstract:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from Tobacco Etch Virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.
