Highly efficient overexpression and purification of multisubunit tethering complexes in Saccharomyces cerevisiae.
Protein Expr Purif
; 212: 106351, 2023 12.
Article
in En
| MEDLINE
| ID: mdl-37574178
ABSTRACT
Vesicle trafficking is a fundamental cellular process that ensures proper material exchange between organelles in eukaryotic cells, and multisubunit tethering complexes (MTCs) are essential in this process. The heterohexameric homotypic fusion and protein sorting (HOPS) complex, which functions in the endolysosomal pathway, is a member of MTCs. Despite its critical role, the complex composition and low-expression level of HOPS have made its expression and purification extremely challenging. In this study, we present a highly efficient strategy for overexpressing and purifying HOPS from Saccharomyces cerevisiae. We achieved HOPS overexpression by integrating a strong promoter TEF1 before each subunit using the gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) system. The HOPS complex was subsequently purified using Staphylococcus aureus protein A (ProtA) affinity purification and size-exclusion chromatography, resulting in high purity and homogeneity. We obtained two-fold more HOPS using this method than that obtained using the commonly used GAL1 promoter-controlled HOPS overexpression. Negative staining electron microscopy analysis confirmed the correct assembly of HOPS. Notably, we also successfully purified two other MTCs, class C core vacuole/endosome tethering (CORVET) and Golgi-associated retrograde protein (GARP) using this approach. Our findings facilitate further in vitro biochemical characterization and functional studies of MTCs and provide a useful guide for the preparation of other heterogenic multisubunit complexes.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Saccharomyces cerevisiae
/
Saccharomyces cerevisiae Proteins
Language:
En
Journal:
Protein Expr Purif
Journal subject:
BIOLOGIA MOLECULAR
Year:
2023
Type:
Article