کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2040028 1073094 2015 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells
ترجمه فارسی عنوان
یک روش مبتنی بر ویرایش مبتنی بر ژنوم برای نقشه برداری مجتمع های چند پروتئینی در سلول های انسانی
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک علوم کشاورزی و بیولوژیک (عمومی)
چکیده انگلیسی


• Streamlined affinity purification of protein complexes using CRISPR/Cas9 and TALENs
• Purification of native holoenzymes regulated under physiological conditions
• Blueprint for the systematic and unbiased mapping of the human protein interactome

SummaryConventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: - Volume 13, Issue 3, 20 October 2015, Pages 621–633
نویسندگان
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