کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843767 | 1069292 | 2005 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (⩾90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (⩾95% purity) of the â¼70 kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises â¼3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly α-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts (â¼100 μg) that make it feasible to consider structural studies of a member of the Na+- and Clâ-dependent neurotransmitter transporter family.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 41, Issue 2, June 2005, Pages 393-401
Journal: Protein Expression and Purification - Volume 41, Issue 2, June 2005, Pages 393-401
نویسندگان
Mark West, Daniel Park, Joanna R. Dodd, Joerg Kistler, David L. Christie,