Simultaneous EGFP and tag labeling of the ß7 subunit for live imaging and affinity purification of functional human proteasomes.
Mol Biotechnol
; 57(1): 36-44, 2015 Jan.
Article
en En
| MEDLINE
| ID: mdl-25164490
ABSTRACT
The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the ß7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the ß7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to ß7-EGFP-HTBH within the proteasome complex rather than to free protein. The ß7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cromatografía de Afinidad
/
Complejo de la Endopetidasa Proteasomal
/
Proteínas Fluorescentes Verdes
/
Imagen Molecular
Límite:
Humans
Idioma:
En
Revista:
Mol Biotechnol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2015
Tipo del documento:
Article
País de afiliación:
Rusia