The synthesis of recombinant membrane proteins in yeast for structural studies

Sarah Routledge; Lina Mikaliunaite; Anjana Patel; Michelle Clare; Stephanie P. Cartwright; Zharain Bawa; Martin D.B. Wilks; Floren Low; David Hardy; Alice J. Rothnie; Roslyn M. Bill

doi:10.1016/j.ymeth.2015.09.027

The synthesis of recombinant membrane proteins in yeast for structural studies

Sarah Routledge, Lina Mikaliunaite, Anjana Patel, Michelle Clare, Stephanie P. Cartwright, Zharain Bawa, Martin D.B. Wilks, Floren Low, David Hardy, Alice J. Rothnie, Roslyn M. Bill^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

Original language	English
Pages (from-to)	26-37
Number of pages	12
Journal	Methods
Volume	95
Early online date	30 Sept 2015
DOIs	https://doi.org/10.1016/j.ymeth.2015.09.027
Publication status	Published - 15 Feb 2016

Bibliographical note

Funding: BBSRC (grants BB/I019960/1, BB/K013319/1, BB/L502194/1 and BB/L015846/1; and the Innovative Medicines Joint Undertaking (grant 115583 to the ND4BB ENABLE Consortium)

Keywords

membrane proteins
yeast
recombinant antigen
expression plasmid vector
promoter
SMALP
cholesterol

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Synthesis of recombinant membrane proteins in yeast
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title = "The synthesis of recombinant membrane proteins in yeast for structural studies",

abstract = "Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.",

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note = "Funding: BBSRC (grants BB/I019960/1, BB/K013319/1, BB/L502194/1 and BB/L015846/1; and the Innovative Medicines Joint Undertaking (grant 115583 to the ND4BB ENABLE Consortium)",

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AU - Mikaliunaite, Lina

AU - Patel, Anjana

AU - Clare, Michelle

AU - Cartwright, Stephanie P.

AU - Bawa, Zharain

AU - Wilks, Martin D.B.

AU - Low, Floren

AU - Hardy, David

AU - Rothnie, Alice J.

AU - Bill, Roslyn M.

N1 - Funding: BBSRC (grants BB/I019960/1, BB/K013319/1, BB/L502194/1 and BB/L015846/1; and the Innovative Medicines Joint Undertaking (grant 115583 to the ND4BB ENABLE Consortium)

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N2 - Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

AB - Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

KW - membrane proteins

KW - yeast

KW - recombinant antigen

KW - expression plasmid vector

KW - promoter

KW - SMALP

KW - cholesterol

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VL - 95

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EP - 37

JO - Methods

JF - Methods

ER -

The synthesis of recombinant membrane proteins in yeast for structural studies

Abstract

Bibliographical note

Keywords

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