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Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Authors

  • Alla Korepanova,

    1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA
    2. National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA
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  • Fei P. Gao,

    1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA
    2. National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA
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  • Yuanzi Hua,

    1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA
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  • Huajun Qin,

    1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA
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  • Robert K. Nakamoto,

    1. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA
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  • Timothy A. Cross

    Corresponding author
    1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA
    2. National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA
    3. Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA
    • National High Magnetic Field Laboratory, 1800 E. Paul Dirac Drive, Tallahassee, FL 32310, USA; fax: (850) 644-1366.
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Abstract

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.

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