Genetic selection system for improving recombinant membrane protein expression in E. coli

Authors

  • Elizabeth Massey-Gendel,

    1. Department of Chemistry and Biochemistry, University of California, Los Angeles, California
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  • Anni Zhao,

    1. Department of Chemistry and Biochemistry, University of California, Los Angeles, California
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  • Gabriella Boulting,

    1. Department of Chemistry and Biochemistry, University of California, Los Angeles, California
    Current affiliation:
    1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
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  • Hye-Yeon Kim,

    1. UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, California
    Current affiliation:
    1. Center for Magnetic Resonance Research, Korea Basic Science Institute, 113 Gwahangno, Yusung-gu, Daejeon, 305-333, Korea
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  • Michael A. Balamotis,

    1. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California
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  • Len M. Seligman,

    1. Department of Biology, Pomona College, Claremont, California
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  • Robert K. Nakamoto,

    1. Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, Virginia
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  • James U. Bowie

    Corresponding author
    1. Department of Chemistry and Biochemistry, University of California, Los Angeles, California
    2. UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, California
    • Boyer Hall, University of California, Los Angeles, 611 Charles E. Young Dr. E, Los Angeles, CA 90095-1570
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Abstract

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

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