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Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3.

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.

SUBMITTER: Diwanji D

PROVIDER: S-EPMC9288109 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3.

Diwanji Devan D Trenker Raphael R Jura Natalia N Verba Kliment A KA

Methods in enzymology 20220405

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 expressio ...[more]

PMID: 35525556

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