SARS-CoV-2 3a expression, purification, and reconstitution into lipid nanodiscs.

Kern, David M; Brohawn, Stephen G

Kern, David M; Brohawn, Stephen G.

Kern DM; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States; Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, United States; California Institute for Quantitative Biology (QB3), University of California Berkeley, Berkeley, CA, United States.
Brohawn SG; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States; Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, United States; California Institute for Quantitative Biology (QB3), University of California Berkeley, Berkeley, CA, United States. Electronic address: brohawn@berkeley.edu.

Methods Enzymol ; 653: 207-235, 2021.

Article in English | MEDLINE | ID: covidwho-1051391

ABSTRACT

ABSTRACT

The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.

Subject(s)

Ion Channels/chemistry; Nanostructures; SARS-CoV-2/chemistry; Viral Envelope Proteins/chemistry; Viroporin Proteins/chemistry; Lipid Bilayers/chemistry

Keywords

Insect cell expression; Ion channel; Membrane protein purification; Membrane protein reconstitution; Nanodisc; SARS-CoV-2

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Viral Envelope Proteins / Nanostructures / Viroporin Proteins / SARS-CoV-2 / Ion Channels Type of study: Observational study / Prognostic study Language: English Journal: Methods Enzymol Year: 2021 Document Type: Article Affiliation country: Bs.mie.2020.12.020

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