کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020359 | 1542331 | 2015 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
We report the protocol for heterologous expression and purification of the N-terminal regulatory region of two Protein Kinase C (PKC)1 isozymes, one conventional and one novel. Previous studies of these domains relied almost exclusively on the fusion constructs with high-molecular-weight solubility fusion partners such as GST and MBP. We developed experimental procedures that enabled us to overcome challenges associated with the amphiphilic character of the regulatory domain and generate sufficient quantities of fusion partner-free proteins for biophysical work. The key features of the protocol are the identity of the cleavable fusion partner, expression conditions, growth medium additives, introduction of mutation/solubility tags, and incorporation of osmolytes. The protein yields are sufficient for cost-effective production of isotopically enriched proteins for NMR work and biophysical studies in general. Our work opens up an avenue for the structural studies of these challenging proteins with high amphiphilic character.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 110, June 2015, Pages 14-21
Journal: Protein Expression and Purification - Volume 110, June 2015, Pages 14-21
نویسندگان
Taylor R. Cole, Tatyana I. Igumenova,