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Chemical Synthesis and Expression of the HIV-1 Rev Protein

Authors

  • Peter Siman,

    1. Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105 (Israel), Fax: (+972) 8-6472943
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    • These authors contributed equally to this work.

  • Ofrah Blatt,

    1. Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904 (Israel)
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    • These authors contributed equally to this work.

  • Tal Moyal,

    1. Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105 (Israel), Fax: (+972) 8-6472943
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  • Dr. Tsafi Danieli,

    1. The Protein Expression and Protein Purification Facilities, Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904 (Israel)
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  • Dr. Mario Lebendiker,

    1. The Protein Expression and Protein Purification Facilities, Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904 (Israel)
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  • Prof. Hilal A. Lashuel,

    1. Laboratory of Molecular Neurobiology and Neuroproteomics, Swiss Federal Institute of Technology Lausanne (EPFL), FSV-BMI AI 2137.1 Station 15, 1015 Lausanne (Switzerland)
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  • Prof. Assaf Friedler,

    Corresponding author
    1. Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904 (Israel)
    • Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904 (Israel)
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  • Prof. Ashraf Brik

    Corresponding author
    1. Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105 (Israel), Fax: (+972) 8-6472943
    • Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105 (Israel), Fax: (+972) 8-6472943
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Abstract

The HIV-1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV-1 lifecycle, thus making Rev an attractive target for the design of anti-HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self-assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one-pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.

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