کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7613547 | 1493589 | 2014 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases
ترجمه فارسی عنوان
پاکسازی مجتمع های پروتئینی از استئوشیومتری زیر واحد را با استفاده از مجموعه ای از پروتئین های متعامد
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
تگ وابستگی، برچسب برداشتن پروتئاز، برش ستون، پروتئین پیچیده، استئوکیومتری واحد تحت کنترل،
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional multi-step chromatography from native or recombinant tag-free sources. Alternatively, they can be expressed and purified using a cleavable N-terminal affinity tag that is subsequently removed by a site-specific protease. Proteolytic tag-removal can also be performed “on-column”. We show here that this not only represents a very efficient workflow, but also drastically improves the purity of the resulting protein preparations. Precondition for effective on-column-cleavage is, however, that the tag-cleaving protease does not bind the stationary phase. We introduce scAtg4 and xlUsp2 as very good and bdSENP1, bdNEDP1 as well as ssNEDP1 as ideal proteases for on-column cleavage at 4 °C. Four of these proteases (bdSENP1, bdNEDP1, scAtg4, xlUsp2) as well as TEV protease display orthogonal, i.e. mutually exclusive cleavage specificities. We combined these features into a streamlined method for the production of highly pure protein complexes: Orthogonal affinity tags and protease recognitions modules are fused to individual subunits. Following co-expression or in-vitro complex assembly, consecutive cycles of affinity capture and proteolytic release then select sequentially for the presence of each orthogonally tagged subunit, yielding protein complexes of well-defined subunit stoichiometry.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1337, 11 April 2014, Pages 106-115
Journal: Journal of Chromatography A - Volume 1337, 11 April 2014, Pages 106-115
نویسندگان
Steffen Frey, Dirk Görlich,