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

doi:10.1007/978-1-0716-0724-4_1

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)

Drug Discovery Unit

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

2 Citations (Scopus)

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 language	English
Title of host publication	Biophysics of Membrane Proteins
Subtitle of host publication	Methods and Protocols
Editors	Vincient L. G. Postis, Adrian Goldman
Place of Publication	New York
Publisher	Humana Press
Pages	3-49
Number of pages	47
Edition	1
ISBN (Electronic)	9781071607244
ISBN (Print)	9781071607237 (hbk), 9781071607268 (pbk)
DOIs	https://doi.org/10.1007/978-1-0716-0724-4_1
Publication status	Published - 2020

Publication series

Name	Methods in Molecular Biology
Volume	2168
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Membrane protein
Protein expression
Protein purification
Bacterial expression
Baculovirus-infected insect cells
Affinity chromatography
Green fluorescence protein
In-gel fluorescence

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10.1007/978-1-0716-0724-4_1

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Liu, Y., Pavić, A., Farley, J. T., de Marcos Lousa , C., Goldman, A., & Postis, V. L. G. (2020). 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 and Protocols (1 ed., pp. 3-49). (Methods in Molecular Biology; Vol. 2168). Humana Press. https://doi.org/10.1007/978-1-0716-0724-4_1

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title = "Membrane protein production and purification from Escherichia coli and Sf9 insect cells",

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.",

keywords = "Membrane protein, Protein expression, Protein purification, Bacterial expression, Baculovirus-infected insect cells, Affinity chromatography, Green fluorescence protein, In-gel fluorescence",

author = "Yixin Liu and Ana Pavi{\'c} and Farley, {Joshua T.} and {de Marcos Lousa}, Carine and Adrian Goldman and Postis, {Vincent L. G.}",

note = "Funding Information: Biotechnology and Biological Sciences Research Council (BBSRC, BB/M021610/1) to AG, https://www.bbsrc.ac.uk/. We would like to thank Steve Baldwin, who continues to be an inspiration in the field of membrane protein research. Copyright: {\textcopyright} 2020 Springer Science+Business Media, LLC, part of Springer Nature.",

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doi = "10.1007/978-1-0716-0724-4_1",

language = "English",

isbn = "9781071607237 (hbk)",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

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Liu, Y, Pavić, A, Farley, JT, de Marcos Lousa , C, Goldman, A & Postis, VLG 2020, Membrane protein production and purification from Escherichia coli and Sf9 insect cells. in VLG Postis & A Goldman (eds), Biophysics of Membrane Proteins: Methods and Protocols. 1 edn, Methods in Molecular Biology, vol. 2168, Humana Press, New York, pp. 3-49. https://doi.org/10.1007/978-1-0716-0724-4_1

Membrane protein production and purification from Escherichia coli and Sf9 insect cells. / Liu, Yixin; Pavić, Ana; Farley, Joshua T. et al.
Biophysics of Membrane Proteins: Methods and Protocols. ed. / Vincient L. G. Postis; Adrian Goldman. 1. ed. New York: Humana Press, 2020. p. 3-49 (Methods in Molecular Biology; Vol. 2168).

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

TY - CHAP

T1 - Membrane protein production and purification from Escherichia coli and Sf9 insect cells

AU - Liu, Yixin

AU - Pavić, Ana

AU - Farley, Joshua T.

AU - de Marcos Lousa , Carine

AU - Goldman, Adrian

AU - Postis, Vincent L. G.

N1 - Funding Information: Biotechnology and Biological Sciences Research Council (BBSRC, BB/M021610/1) to AG, https://www.bbsrc.ac.uk/. We would like to thank Steve Baldwin, who continues to be an inspiration in the field of membrane protein research. Copyright: © 2020 Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - Membrane protein

KW - Protein expression

KW - Protein purification

KW - Bacterial expression

KW - Baculovirus-infected insect cells

KW - Affinity chromatography

KW - Green fluorescence protein

KW - In-gel fluorescence

UR - http://europepmc.org/abstract/med/33582985

U2 - 10.1007/978-1-0716-0724-4_1

DO - 10.1007/978-1-0716-0724-4_1

M3 - Chapter (peer-reviewed)

C2 - 33582985

SN - 9781071607237 (hbk)

SN - 9781071607268 (pbk)

T3 - Methods in Molecular Biology

SP - 3

EP - 49

BT - Biophysics of Membrane Proteins

A2 - Postis, Vincient L. G.

A2 - Goldman, Adrian

PB - Humana Press

CY - New York

ER -

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

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