Membrane protein production and purification from Escherichia coli and Sf9 insect cells.

Yixin Liu, Ana Pavić, Joshua T. Farley, Carine de Marcos Lousa , Adrian Goldman, Vincent L.G. Postis (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

Abstract

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
Original languageUndefined/Unknown
Title of host publicationBiophysics of Membrane Proteins
Subtitle of host publicationMethods and Protocols
EditorsVincient L.G. Postis, Adrian Goldman
PublisherHumana Press
Pages3-49
Number of pages47
Edition1
ISBN (Electronic)978-1-0716-0724-4
ISBN (Print)978-1-0716-0723-7
DOIs
Publication statusPublished - 1 Jan 2020

Publication series

NameMethods in Molecular Biology (Clifton, N.J.)
PublisherSpringer Verlag
ISSN (Print)1064-3745

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