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

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

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.

Key words

Membrane protein Green fluorescent protein (GFP) Titratable T7 RNA polymerase Riboswitch RiboTite T7 lysozyme 

Notes

Acknowledgments

The Oxford Protein Production Facility-UK is supported by the UK Medical Research Council (MR/K018779/1) and the Manchester Group are supported by the Biotechnology and Biology Research Council, ND holds a BBSRC David Phillips Fellowship [BB/K014773/1].

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Manchester Institute of BiotechnologyUniversity of ManchesterManchesterUK
  2. 2.Research Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  3. 3.School of BiosciencesUniversity of NottinghamLoughborough, LeicestershireUK
  4. 4.Oxford Protein Production Facility-UK, Research Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  5. 5.Division of Structural Biology, Henry Wellcome Building for Genomic MedicineUniversity of OxfordRoosevelt Drive, OxfordUK

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