کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7233494 | 1470972 | 2014 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system
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کلمات کلیدی
E. coliIndium tinoxideTCADPhPCInositol trisphosphateIP3GPCRDNA - DNA یا اسید دزوکسی ریبونوکلئیکG-protein coupled receptor - G-پروتئین همراه گیرندهKcsA - KCSASDS–PAGE - SDS-PAGEdeoxyribonucleic acid - اسید deoxyribonucleictricholoroacetic acid - اسید تریکولورواتیکEscherichia coli - اشریشیا کُلیsodium dodecyl sulfate–polyacrylamide gel electrophoresis - الکتروفورز ژل دوده سولفات سدیم پلی آکریل آمیدITO - اینCell-free - بدون سلولMembrane protein expression - بیان پروتئین غشاءSingle-channel current - جریان تک کانالMicrosome - میکروسومpolymerase chain reaction - واکنش زنجیره ای پلیمرازPCR - واکنش زنجیرهٔ پلیمرازIon channel - کانال یونی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system](/preview/png/7233494.png)
چکیده انگلیسی
The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90Â min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biosensors and Bioelectronics - Volume 59, 15 September 2014, Pages 174-183
Journal: Biosensors and Bioelectronics - Volume 59, 15 September 2014, Pages 174-183
نویسندگان
Srujan Kumar Dondapati, Mohamed Kreir, Robert B. Quast, Doreen A Wüstenhagen, Andrea Brüggemann, Niels Fertig, Stefan Kubick,