Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

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Liu , Y , Pavic , A , Farley , J T , Lousa , C D M , Goldman , A & Postis , V L G 2021 , Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells . in V L G Postis & A Goldman (eds) , Biophysics of Membrane Proteins . Methods in Molecular Biology , vol. 2168 , Humana , pp. 3-49 . https://doi.org/10.1007/978-1-0716-0724-4_1

Title: Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells
Author: Liu, Yixin; Pavic, Ana; Farley, Joshua T.; Lousa, Carine De Marcos; Goldman, Adrian; Postis, Vincent L. G.
Other contributor: Postis, Vincent L. G.
Goldman, Adrian
Contributor organization: Molecular and Integrative Biosciences Research Programme
Biochemistry and Biotechnology
Publisher: Humana
Date: 2021
Language: eng
Number of pages: 47
Belongs to series: Biophysics of Membrane Proteins
Belongs to series: Methods in Molecular Biology
ISBN: 978-1-0716-0723-7
978-1-0716-0724-4
ISSN: 1940-6029
DOI: https://doi.org/10.1007/978-1-0716-0724-4_1
URI: http://hdl.handle.net/10138/340196
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.
Subject: 1182 Biochemistry, cell and molecular biology
Membrane protein
Protein expression
Protein purification
Bacterial expression
Baculovirus-infected insect cells
Affinity chromatography
Green fluorescence protein
In-gel fluorescence
HIGH-LEVEL EXPRESSION
AFFINITY-CHROMATOGRAPHY
RECOMBINANT PROTEINS
CRYSTAL-STRUCTURE
CARRIER PROTEINS
BACULOVIRUS DNA
FUSION SYSTEMS
SCALE-UP
OVEREXPRESSION
VECTORS
Peer reviewed: Yes
Rights: unspecified
Usage restriction: openAccess
Self-archived version: acceptedVersion


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