title: Functional analysis and purification of a Pen-2 fusion protein for γ-secretase structural studies.
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.
authors.affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Holmes
authors.forename: Swetha
authors.initials: S
authors.lastname: Paturi
authors.forename: Michael S
authors.initials: MS
authors.lastname: Wolfe
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Selkoe
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout
chemical_list: D008565:Membrane Proteins; C469349:PSENEN protein, human; D053829:Amyloid Precursor Protein Secretases
keywords: Alzheimer's disease; Pen-2; γ-secretase