Optimization of Membrane Protein Production Using Titratable Strains of E. coli

Rosa Morra; Kate Young; David  Casas-Mao; Neil Dixon; Louise E Bird

doi:10.1007/978-1-4939-6887-9_6

Optimization of Membrane Protein Production Using Titratable Strains of E. coli

Rosa Morra, Kate Young, David Casas-Mao, Neil Dixon, Louise E Bird

Research output: Chapter in Book/Conference proceeding › Chapter › peer-review

Abstract

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

Original language	English
Title of host publication	Heterologous Gene Expression in E.coli
Editors	Nicola A. Burgess-Brown
Publisher	Springer Nature
Pages	83-107
Number of pages	25
ISBN (Electronic)	9781493968879
ISBN (Print)	9781493968855
DOIs	https://doi.org/10.1007/978-1-4939-6887-9_6
Publication status	Published - 2017

Publication series

Name	Methods in Molecular Biology
Volume	1586

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10.1007/978-1-4939-6887-9_6

Development and Application of Next Generation Synthetic Biology Tools
Dixon, N. (PI)
1/11/13 → 31/10/19
Project: Research

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abstract = "The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.",

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AU - Bird, Louise E

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AB - The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

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ER -

Optimization of Membrane Protein Production Using Titratable Strains of E. coli

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Development and Application of Next Generation Synthetic Biology Tools

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