Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter

  • Ekaitz Errasti-Murugarren
  • Arturo Rodríguez-Banqueri
  • José Luis Vázquez-Ibar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library’s members.

Key words

Split GFP Membrane transport proteins Heterologous expression SteT LAT FSEC 

Notes

Acknowledgments

We are very grateful to Geoffrey S. Waldo and members of his lab for sending us the split GFP expression plasmids. We also thank Manuel Palacín for his support and critical advice. This work was supported in part by the Spanish Ministry of Science and Innovation, grants BFU2008-04637 (J.L.V.I.). E. Errasti-Murugarren is a recipient of a Sara Borrell contract from the Instituto de Salud Carlos III (Spain). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). The authors declare no competing financial interests.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ekaitz Errasti-Murugarren
    • 1
  • Arturo Rodríguez-Banqueri
    • 1
    • 2
  • José Luis Vázquez-Ibar
    • 1
    • 3
  1. 1.Institute for Research in Biomedicine (IRB Barcelona)Barcelona Institute of Science and TechnologyBarcelonaSpain
  2. 2.Unitat de Proteòmica Aplicada i Enginyeria de Proteïnes, Institut de Biotecnologia i Biomedicina (IBB)Universitat Autònoma de Barcelona (UAB)BarcelonaSpain
  3. 3.Institute for Integrative Biology of the Cell (I2BC), iBiTec-S/SB2SM, CEA Saclay CNRS UMR 9198, University Paris-SudUniversity Paris-SaclayGif-sur-Yvette CedexFrance

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