Proteinase K goes thermo-labile

Proteinase K, originally from the fungus Tritirachium album, is a highly active serine protease with broad cleavage specificity. This enzyme is widely used to remove proteins/enzymes in nucleic acid samples. However, use of wildtype proteinase K (WTPK) in multi-step enzymatic workflows such as next...

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Main Authors: Chen, Minyong, Shi, Stephen, Paschal, Beth, Bielik, Alicia, Sakhtah, Hassan, Guthrie, Ellen, Causey, Bryce, Samuelson, James C
Format: Text
Language:unknown
Published: ECI Digital Archives 2019
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Online Access:https://dc.engconfintl.org/biochem_xxi/36
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spelling fteci:oai:dc.engconfintl.org:biochem_xxi-1091 2023-05-15T15:16:46+02:00 Proteinase K goes thermo-labile Chen, Minyong Shi, Stephen Paschal, Beth Bielik, Alicia Sakhtah, Hassan Guthrie, Ellen Causey, Bryce Samuelson, James C 2019-07-15T07:00:00Z https://dc.engconfintl.org/biochem_xxi/36 unknown ECI Digital Archives https://dc.engconfintl.org/biochem_xxi/36 Biochemical and Molecular Engineering XXI Proteinase K Thermolabile Next Generation Sequencing NGS workflow Heat Labile Biochemical and Biomolecular Engineering Engineering text 2019 fteci 2022-12-27T14:51:24Z Proteinase K, originally from the fungus Tritirachium album, is a highly active serine protease with broad cleavage specificity. This enzyme is widely used to remove proteins/enzymes in nucleic acid samples. However, use of wildtype proteinase K (WTPK) in multi-step enzymatic workflows such as next generation sequencing (NGS) is limited due to its extreme thermostability and ineffective removal by heat treatment. The purpose of this study was to engineer a thermolabile Proteinase K (TLPK) as active as WTPK, which may be fully inactivated at 65°C or below to minimize DNA/RNA damage. Using molecular engineering approaches, we have successfully obtained TLPK. As shown in Figure 1, TLPK is almost as active as WTPK at 37°C using native bovine serum albumin (BSA) as substrate. Importantly, TLPK can be efficiently inactivated within the temperature range of 55°C to 65°C, which is demonstrated by loss of protease activity on bovine serum albumin (BSA) substrate (Figure 2a) and a colorimetric peptide substrate (Figure 2b) after heat treatment. Compared to WTPK, TLPK shows over 20°C more labile to heat inactivation. The melting temperature (Tm) of TLPK is also around 25°C lower than that of WTPK, decreasing from 75.9°C to 50.9°C. TLPK greatly outperforms a broad specificity protease isolated from an arctic marine microbial source, both by specific enzyme activity and thermolability. One of the TLPK applications is it can inactivate heat resistant restriction enzymes such as PvuII and PstI without affecting downstream reactions. The mainstream applications may be its incorporation into multi-step enzymatic workflows such as NGS sample preparation. Unlike WTPK, TLPK can be used to eliminate an enzyme function without contaminating the next enzymatic step in the same reaction vessel. New England Biolabs has tested TLPK and found it to simplify and improve NGS workflows. Please click Additional Files below to see the full abstract. Text Arctic Engineering Conferences International: ECI Digital Archives Arctic
institution Open Polar
collection Engineering Conferences International: ECI Digital Archives
op_collection_id fteci
language unknown
topic Proteinase K
Thermolabile
Next Generation Sequencing
NGS workflow
Heat Labile
Biochemical and Biomolecular Engineering
Engineering
spellingShingle Proteinase K
Thermolabile
Next Generation Sequencing
NGS workflow
Heat Labile
Biochemical and Biomolecular Engineering
Engineering
Chen, Minyong
Shi, Stephen
Paschal, Beth
Bielik, Alicia
Sakhtah, Hassan
Guthrie, Ellen
Causey, Bryce
Samuelson, James C
Proteinase K goes thermo-labile
topic_facet Proteinase K
Thermolabile
Next Generation Sequencing
NGS workflow
Heat Labile
Biochemical and Biomolecular Engineering
Engineering
description Proteinase K, originally from the fungus Tritirachium album, is a highly active serine protease with broad cleavage specificity. This enzyme is widely used to remove proteins/enzymes in nucleic acid samples. However, use of wildtype proteinase K (WTPK) in multi-step enzymatic workflows such as next generation sequencing (NGS) is limited due to its extreme thermostability and ineffective removal by heat treatment. The purpose of this study was to engineer a thermolabile Proteinase K (TLPK) as active as WTPK, which may be fully inactivated at 65°C or below to minimize DNA/RNA damage. Using molecular engineering approaches, we have successfully obtained TLPK. As shown in Figure 1, TLPK is almost as active as WTPK at 37°C using native bovine serum albumin (BSA) as substrate. Importantly, TLPK can be efficiently inactivated within the temperature range of 55°C to 65°C, which is demonstrated by loss of protease activity on bovine serum albumin (BSA) substrate (Figure 2a) and a colorimetric peptide substrate (Figure 2b) after heat treatment. Compared to WTPK, TLPK shows over 20°C more labile to heat inactivation. The melting temperature (Tm) of TLPK is also around 25°C lower than that of WTPK, decreasing from 75.9°C to 50.9°C. TLPK greatly outperforms a broad specificity protease isolated from an arctic marine microbial source, both by specific enzyme activity and thermolability. One of the TLPK applications is it can inactivate heat resistant restriction enzymes such as PvuII and PstI without affecting downstream reactions. The mainstream applications may be its incorporation into multi-step enzymatic workflows such as NGS sample preparation. Unlike WTPK, TLPK can be used to eliminate an enzyme function without contaminating the next enzymatic step in the same reaction vessel. New England Biolabs has tested TLPK and found it to simplify and improve NGS workflows. Please click Additional Files below to see the full abstract.
format Text
author Chen, Minyong
Shi, Stephen
Paschal, Beth
Bielik, Alicia
Sakhtah, Hassan
Guthrie, Ellen
Causey, Bryce
Samuelson, James C
author_facet Chen, Minyong
Shi, Stephen
Paschal, Beth
Bielik, Alicia
Sakhtah, Hassan
Guthrie, Ellen
Causey, Bryce
Samuelson, James C
author_sort Chen, Minyong
title Proteinase K goes thermo-labile
title_short Proteinase K goes thermo-labile
title_full Proteinase K goes thermo-labile
title_fullStr Proteinase K goes thermo-labile
title_full_unstemmed Proteinase K goes thermo-labile
title_sort proteinase k goes thermo-labile
publisher ECI Digital Archives
publishDate 2019
url https://dc.engconfintl.org/biochem_xxi/36
geographic Arctic
geographic_facet Arctic
genre Arctic
genre_facet Arctic
op_source Biochemical and Molecular Engineering XXI
op_relation https://dc.engconfintl.org/biochem_xxi/36
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