Proteinase K goes thermo-labile
July 14-18, 2019
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.
Minyong Chen, Stephen Shi, Beth Paschal, Alicia Bielik, Hassan Sakhtah, Ellen Guthrie, Bryce Causey, and James C. Samuelson, "Proteinase K goes thermo-labile" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/36