Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3.
Methods Enzymol
; 667: 611-632, 2022.
Article
in En
| MEDLINE
| ID: mdl-35525556
ABSTRACT
Biochemical analyses of membrane receptor kinases have been limited by challenges in obtaining sufficient homogeneous receptor samples for downstream structural and biophysical characterization. Here, we report a suite of methods for the efficient expression, purification, and visualization by cryo-electron microscopy (cryo-EM) of near full-length Human Epidermal Growth Factor Receptor 3 (HER3), a receptor tyrosine pseudokinase, in the unliganded state. Through transient mammalian cell expression, a two-step purification with detergent exchange into lauryl maltose neopentyl glycol (LMNG), and freezing devoid of background detergent micelle, we obtained ~6Å reconstructions of the ~60kDa fully-glycosylated unliganded extracellular domain of HER3 from just 30mL of suspension culture. The reconstructions reveal previously unappreciated extracellular domain dynamics and glycosylation sites.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Detergents
/
Micelles
Limits:
Animals
/
Humans
Language:
En
Journal:
Methods Enzymol
Year:
2022
Document type:
Article
Affiliation country:
United States