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
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
نویسندگان
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