Functional analysis and purification of a Pen-2 fusion protein for γ-secretase structural studies

Authors

  • Oliver Holmes,

    1. Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA
    2. Harvard Medical School, Boston, MA, USA
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  • Swetha Paturi,

    1. Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA
    2. Harvard Medical School, Boston, MA, USA
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  • Michael S. Wolfe,

    1. Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA
    2. Harvard Medical School, Boston, MA, USA
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  • Dennis J. Selkoe

    Corresponding author
    1. Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA
    2. Harvard Medical School, Boston, MA, USA
    • Address correspondence and reprint requests to Dennis J. Selkoe, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Harvard Institutes of Medicine, Room 730, Boston, MA 02115, USA. E-mail: dselkoe@rics.bwh.harvard.edu

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Abstract

The 19-transmembrane, multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal maltose binding protein tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex.

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We present a method by which an MBP-tagged form of the Pen-2 subunit of γ-secretase may be purified from bacteria to mg quantities at greater than 95% purity. We show that this construct is incorporated into and allows full activity of γ-secretase in a mammalian system. These methods provide valuable tools to study the structure and function of Pen-2 and γ-secretase.

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