Efficient Expression, Purification, and Visualization by Cryo-EM of Unliganded Near Full-length HER3

Overview

Journal Methods Enzymol

Specialty Biochemistry

Date 2022 May 7

PMID 35525556

Authors

Devan Diwanji

Raphael Trenker

Natalia Jura

Kliment A Verba

Affiliations

Soon will be listed here.

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.

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