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

Holmes O1, Paturi S, Wolfe MS, Selkoe DJ.
Source: J Neurochem
Publication Date: (2014)
Issue: 131 (1): 94-100
Research Area:
Cells used in publication:
Embryonic fibroblast, mouse (MEF) immort
Species: mouse
Tissue Origin: embryo
4D-Nucleofector® X-Unit
Pen-2 knockout mouse embryonic fibroblast cell line (immortalized with large T-Antigen) was transfected using the 4D-Nucleofector X Unit and P3 Primary Cell 4D-Nucleofector X Kit, recovered in RPMI media + 10% FBS. Six hours after plating, media were replaced with fresh DMEM + 10% FBS.
The 19-transmembrane, multisubunit ?-secretase complex generates the amyloid ß-peptide (Aß) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the ß-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 ?-secretase complex. We present a method by which an MBP-tagged form of the Pen-2 subunit of ?-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 ?-secretase in a mammalian system. These methods provide valuable tools to study the structure and function of Pen-2 and ?-secretase.