{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T20:07:04Z","timestamp":1773173224747,"version":"3.50.1"},"reference-count":26,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2014,6,18]],"date-time":"2014-06-18T00:00:00Z","timestamp":1403049600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["P01 AG015379"],"award-info":[{"award-number":["P01 AG015379"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Journal of Neurochemistry"],"published-print":{"date-parts":[[2014,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The 19\u2010transmembrane, multisubunit \u03b3\u2010secretase complex generates the amyloid \u03b2\u2010peptide (A\u03b2) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the \u03b2\u2010amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen\u20102), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high\u2010resolution 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\u20102 can be purified from bacteria to &gt;\u00a095% 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\u2010terminal maltose binding protein tag on Pen\u20102 still permits incorporation into the complex and subsequent presenilin\u20101 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen\u20102 and the \u03b3\u2010secretase complex.<\/jats:p><jats:p>\n<jats:boxed-text content-type=\"graphic\" position=\"anchor\"><jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" mimetype=\"image\/png\" position=\"anchor\" specific-use=\"enlarged-web-image\" xlink:href=\"graphic\/jnc12772-fig-0008-m.png\"><jats:alt-text>image<\/jats:alt-text><\/jats:graphic><\/jats:boxed-text>\n<\/jats:p><jats:p>We present a method by which an MBP\u2010tagged form of the Pen\u20102 subunit of \u03b3\u2010secretase 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 \u03b3\u2010secretase in a mammalian system. These methods provide valuable tools to study the structure and function of Pen\u20102 and \u03b3\u2010secretase.<\/jats:p>","DOI":"10.1111\/jnc.12772","type":"journal-article","created":{"date-parts":[[2014,5,28]],"date-time":"2014-05-28T08:25:25Z","timestamp":1401265525000},"page":"94-100","source":"Crossref","is-referenced-by-count":3,"title":["Functional analysis and purification of a Pen\u20102 fusion protein for \u03b3\u2010secretase structural studies"],"prefix":"10.1111","volume":"131","author":[{"given":"Oliver","family":"Holmes","sequence":"first","affiliation":[{"name":"Center for Neurologic Diseases Brigham and Women's Hospital Boston MA USA"},{"name":"Harvard Medical School Boston MA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Swetha","family":"Paturi","sequence":"additional","affiliation":[{"name":"Center for Neurologic Diseases Brigham and Women's Hospital Boston MA USA"},{"name":"Harvard Medical School Boston MA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael S.","family":"Wolfe","sequence":"additional","affiliation":[{"name":"Center for Neurologic Diseases Brigham and Women's Hospital Boston MA USA"},{"name":"Harvard Medical School Boston MA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dennis J.","family":"Selkoe","sequence":"additional","affiliation":[{"name":"Center for Neurologic Diseases Brigham and Women's Hospital Boston MA USA"},{"name":"Harvard Medical School Boston MA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,6,18]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1013246107"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.216978"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.62.1.204-229.1998"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb960"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1150755"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/359322a0"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M406289200"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M611549200"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M509070200"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M412404200"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1037392100"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0609981104"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature11801"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2013.09.018"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1111\/jnc.12016"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.10.078"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.2005.03165.x"},{"key":"e_1_2_7_19_1","volume-title":"ImageJ","author":"Rasband W. 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