Expression, purification, and biological activity evaluation of cathepsin L in mammalian cells.
Biosci Biotechnol Biochem
; 88(4): 405-411, 2024 Mar 22.
Article
in En
| MEDLINE
| ID: mdl-38271604
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Spike Glycoprotein, Coronavirus
/
COVID-19
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Biosci Biotechnol Biochem
Journal subject:
BIOQUIMICA
/
BIOTECNOLOGIA
Year:
2024
Document type:
Article
Affiliation country:
China