کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359853 | 1542322 | 2016 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification and guanine nucleotide binding characterization of Arabidopsis RabE1d13-185 GTPase
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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
Arabidopsis RabE1d subclass plays important plant-specific functions in plant growth and development, response to ethylene and defence to plant pathogen, besides their basic cellular role in membrane trafficking. In this study, we present the expression, purification, and characterization of the recombinant core domain of AtRabE1d13-185. AtRabE1d13-185 was successfully expressed in Escherichia coli and purified via two-step nickel affinity chromatography followed by gel filtration, and identified single band in SDS-PAGE. The resultant protein was functionally active, as determined by interaction with guanine nucleotide by a fluorescence-based assay. The intrinsic tryptophan of AtRabE1d13-185 showed fluorescence resonance energy transfer (FRET) effect upon forming complex with fluorescent methylanthraniloyl (mant)-GDP, but quenched when binding with non-labelled guanine nucleotide. The association rate of mantGDP with AtRabE1d13-185 was determined to be 3.48Â ÃÂ 107Â sâ1Â Mâ1. The dissociation rates of GDP and mantGDP from the complex with AtRabE1d13-185 were similar. The koff values were determined to be 4.02Â ÃÂ 10â4Â sâ1 based on the FRET effect for the AtRabE1d13-185:GDP and 5.41Â ÃÂ 10â4Â sâ1 for mantGDP excited directly.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 119, March 2016, Pages 57-62
Journal: Protein Expression and Purification - Volume 119, March 2016, Pages 57-62
نویسندگان
Yi Gao, Lingling Tan, Chun-Hai Dong, Xiaomin Hou,