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A method for producing protease pS273R of the African swine fever virus
Danil S Kalinin1, Sergey G Mayorov2, Marina Yu Zemskova2
1A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russian Federation.
Abstract:
The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.
Insights
African swine fever virus (ASFV) pS273R protease was produced in E. coli using a SlyD chaperone fusion. This method yields soluble, active protease and a new fluorescence assay for its activity.
Area of Science:
- Biochemistry
- Virology
- Molecular Biology
Background:
- The pS273R protease of African swine fever virus (ASFV) is crucial for viral replication, processing polyproteins pp220 and pp62.
- Its essential role makes it a promising target for antiviral drug development.
Purpose of the Study:
- To develop an efficient method for producing active pS273R protease in E. coli.
- To establish a novel fluorescence-based assay for measuring pS273R protease activity.
Main Methods:
- Co-expression of pS273R protease with SlyD chaperone in E. coli to ensure solubility and activity.
- Autocatalytic cleavage of the SlyD fusion protein to release active pS273R.
- Chromatographic purification of the viral protease.
- Development of a fluorescence assay using thioredoxin-EGFP substrate cleavage detection.
Main Results:
- A soluble and enzymatically active pS273R protease was successfully produced in E. coli.
- A straightforward purification protocol yielded highly purified viral protease.
- The developed fluorescence method effectively assesses pS273R activity, demonstrating substrate cleavage even in complex mixtures like cell lysates.
Conclusions:
- The SlyD fusion strategy provides a robust method for producing active ASFV pS273R protease.
- The novel fluorescence assay is a sensitive and selective tool for monitoring pS273R protease activity.
- This work facilitates further research into ASFV protease function and antiviral strategies.
