Temperature Sensitive “V24” and “V25” Protease
Background
Heat inactivation is a common approach used in molecular biology to remove enzyme activity. Subtilisins represent one of the most widely used proteases in molecular biology workflows. Proteinase K is a subtilisin serine protease that is used in isolation of DNA and RNA, inactivation of RNAase, DNAase, PCR purification and removes enzymes from DNA to improve cloning efficiency. Proteinase K activates at 20˚C and is optimized between 50˚C and 65˚C. It is deactivated at 95ﹾC for 10 minutes. Because Proteinase K requires high temperatures for optimal activation and requires temperatures, for an extended period, to deactivate activity it can threaten the integrity of the nucleic acid sample; therefore, it is not suitable for many specific diagnostic and industrial needs.
Overview
Researchers have generated two novel temperature-sensitive (TS) variants of subtilisin Carlsberg (SubC) protease expressed in Bacillus subtilis strain, that we have named Variant 24 (V24) and Variant 25 (V25). The subtilisins are efficiently expressed in a B. subtilis system. The B. subtilis has all seven of its natural protease genes deleted. V24 and V25 are proven to be capable of degrading a broad range of proteins in their native or denatured state. The protease variant V24 has an inactivation profile that allows protease digestion at 40˚C and inactivation at 50˚C. This variant of SubC could be useful when working with a fragile specimen when minimal heat treatment is desired, such as in automatic diagnostic systems. V25 has properties that allow it to digest a substrate at elevated temperatures, such as 55˚C and still be deactivated at a moderate temperature in the 60˚C – 65˚C range. This variant would be useful if one wanted to denature proteins in a complex mixture and still have the option of inactivating the enzyme below 65˚C. There is no need for column purification or a treatment at extreme temperatures to completely deactivate these two variants. V24 and V25 will increase efficiently and improve workflows in molecular biology research, automated processes such as Next‑Gen Sequencing (NGS) and disease diagnostics.
Benefits
- Excellent efficiency, all activity of V24 is destroyed after a 10-minute incubation period at 50˚C; all activity of V25 is destroyed after a 10 minute incubation period at 60°C.
- Activity remains stable over several months when the protease is stored at 4˚C or lower; when stored at -80°C the protease remains stable for up to two years
- Detergent compatibility, protease remains active in the presence of detergents such as SDS and Triton X-100.
- Provides time and cost savings, V24 and V25 eliminates the requirement for a purification step.
Applications
- Molecular biology research
- Medical diagnostics
- Automated processes, such as Next-Gen Sequencing (NGS).
Opportunity
- Technology licensing
- Material transfer agreements
Patents
- Canadian Patent Application No. 3028612
- US Patent No. 10894954