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

David M Kern; Stephen G Brohawn

doi:10.1016/bs.mie.2020.12.020

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

Methods Enzymol. 2021:653:207-235. doi: 10.1016/bs.mie.2020.12.020. Epub 2021 Jan 27.

Authors

David M Kern¹, Stephen G Brohawn²

Affiliations

¹ 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.
² 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.

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.

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

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

3a protein, SARS-CoV
Ion Channels
Lipid Bilayers
Viral Envelope Proteins
Viroporin Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding