کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2022011 | 1069273 | 2007 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor
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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
![عکس صفحه اول مقاله: Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor](/preview/png/2022011.png)
چکیده انگلیسی
The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2 mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an α-helical content of the purified recombinant protein of â¼54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 53, Issue 1, May 2007, Pages 153-163
Journal: Protein Expression and Purification - Volume 53, Issue 1, May 2007, Pages 153-163
نویسندگان
Alexei Yeliseev, Lioudmila Zoubak, Klaus Gawrisch,