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

    Research output: Chapter in BookChapterResearchpeer-review

    2 Citations (Scopus)

    Abstract

    © 2017, Springer Science+Business Media LLC. 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.
    Original languageEnglish
    Title of host publicationMethods in Molecular Biology
    Pages181-195
    Number of pages14
    Volume1586
    DOIs
    Publication statusPublished - 1 Jan 2017

    Keywords

    • FSEC
    • Heterologous expression
    • LAT
    • Membrane transport proteins
    • Split GFP
    • SteT

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