Structural and Functional Characterization of Schistosoma mansoni Cathepsin B1.
Animals
Cathepsin B
/ antagonists & inhibitors
Crystallization
Electroporation
Enzyme Activation
Gene Expression
Genetic Vectors
/ metabolism
Glycosylation
Kinetics
Mutation
/ genetics
Recombinant Proteins
/ isolation & purification
Saccharomycetales
/ genetics
Schistosoma mansoni
/ enzymology
Transformation, Genetic
Activity assay
Cathepsin B
Crystal structure
Expression
Inhibition
Parasite
Protease
Schistosoma mansoni
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
27
5
2020
pubmed:
27
5
2020
medline:
12
3
2021
Statut:
ppublish
Résumé
Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.
Identifiants
pubmed: 32452002
doi: 10.1007/978-1-0716-0635-3_12
doi:
Substances chimiques
Recombinant Proteins
0
Cathepsin B
EC 3.4.22.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM