In:
Journal of Neurochemistry, Wiley, Vol. 131, No. 1 ( 2014-10), p. 94-100
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. image 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.
Type of Medium:
Online Resource
ISSN:
0022-3042
,
1471-4159
DOI:
10.1111/jnc.2014.131.issue-1
Language:
English
Publisher:
Wiley
Publication Date:
2014
detail.hit.zdb_id:
2020528-4
SSG:
12
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