کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5515953 1542299 2018 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Disassembly/reassembly strategy for the production of highly pure GroEL, a tetradecameric supramolecular machine, suitable for quantitative NMR, EPR and mutational studies
کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Disassembly/reassembly strategy for the production of highly pure GroEL, a tetradecameric supramolecular machine, suitable for quantitative NMR, EPR and mutational studies
چکیده انگلیسی


- A straightforward purification protocol involving disassembly and reassembly of GroEL, a dual-ringed tetradecamer, is described.
- Effectively removes naturally occurring, tightly bound proteins present in the bacterial expression system.
- Suitable for isotope labeling, deuteration and introduction of site-specific paramagnetic labels via surface engineered cysteine mutations.
- Provides yields in sufficient quantities suitable for both NMR and EPR studies.

GroEL, a prototypical member of the chaperonin class of chaperones, is a large supramocular machine that assists protein folding and plays an important role in proteostasis. GroEL comprises two heptameric rings, each of which encloses a large cavity that provides a folding chamber for protein substrates. Many questions remain regarding the mechanistic details of GroEL facilitated protein folding. Thus, data at atomic resolution of the type provided by NMR and EPR are invaluable. Such studies often require complete deuteration of GroEL, uniform or residue specific 13C and 15N isotope labeling, and the introduction of selective cysteine mutations for site-specific spin labeling. In addition, high purity GroEL is essential for detailed studies of substrate-GroEL interactions as quantitative interpretation is impossible if the cavities are already occupied and blocked by other protein substrates present in the bacterial expression system. Here we present a new purification protocol designed to provide highly pure GroEL devoid of non-specific protein substrate contamination.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 142, February 2018, Pages 8-15
نویسندگان
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