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Expression, purification, and biological activity evaluation of cathepsin L in mammalian cells
Wenwen Zhou1, Baoqing You1, Yifan Zheng1
1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Abstract:
Cathepsin L (CTSL) could cleave and activate SARS-CoV-2 Spike protein to promote viral entry, making it a hopeful therapeutic target for COVID-19 prevention and treatment. So CTSL inhibitors are considered to be a promising strategy to SARS-CoV-2 infection. CTSL has previously been expressed in inclusion body in Escherichia coli. In order to prepare CTSL with high purity and activity in soluble active form, we transformed HEK-293T cells with a recombinant mammalian expression plasmid. CTSL was purified to a purity about 95%, found to migrate at approximately 43 kDa and exhibited substrate specificity against Z-Phe-Arg-AMC with specific activity of no less than 85 081 U/mg, characteristic of active CTSL. Although eukaryotic purified CTSL is commercially available, our study for the first time reported the details of the expression, purification, and characterization of active, recombinant CTSL in eukaryocyte system, which laid an experimental foundation for the establishment of high-throughput screening model for anti-coronavirus drugs targeting CTSL.
Insights
Researchers developed a method to produce active Cathepsin L (CTSL) in a soluble form using HEK-293T cells. This breakthrough aids in developing new COVID-19 therapies targeting the SARS-CoV-2 virus.
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Cathepsin L (CTSL) is crucial for SARS-CoV-2 entry by activating the Spike protein.
- CTSL inhibitors represent a potential therapeutic strategy against SARS-CoV-2 infection.
- Previous attempts to express CTSL in E. coli resulted in inclusion bodies, hindering soluble active form production.
Purpose of the Study:
- To express and purify active, soluble Cathepsin L (CTSL) in a eukaryotic system.
- To characterize the biochemical properties of the recombinant CTSL.
- To establish a foundation for high-throughput screening of anti-coronavirus drugs targeting CTSL.
Main Methods:
- Transfection of HEK-293T cells with a recombinant mammalian expression plasmid for CTSL.
- Purification of CTSL to high purity.
- Biochemical characterization including SDS-PAGE, substrate specificity, and specific activity assays.
Main Results:
- Successfully expressed and purified active, soluble CTSL with approximately 95% purity.
- Purified CTSL migrated at approximately 43 kDa on SDS-PAGE.
- Demonstrated characteristic substrate specificity and high specific activity (≥85,081 U/mg) against Z-Phe-Arg-AMC.
Conclusions:
- This study provides the first detailed report on the expression, purification, and characterization of active, recombinant CTSL in a eukaryotic system.
- The developed method yields high-purity, active CTSL, overcoming limitations of previous expression systems.
- This work establishes a crucial experimental foundation for developing high-throughput screening models for novel anti-coronavirus drugs targeting CTSL.
