کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10156932 | 1666436 | 2018 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Systematic optimization of cell-free synthesized human endothelin B receptor folding
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کلمات کلیدی
DOPC1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)feeding mixture1,2-dioleoyl-sn-glycero-3-phospho-l-serinePOPGDPPGNanodisc1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)DMPGendothelin receptor type BDOPGETBDOPSDPCMSPET-1SECdMPCDTTGSHGSSGIMAC1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) - 1-پالمیتویل-2-اولئویل-اس-گلیسرو-3-فسفات (1'-rac-glycerol)1,2-dimyristoyl-sn-glycero-3-phosphocholine - 1،2-dimyristoyl-sn-glycero-3-phosphosocholine1,2-dioleoyl-sn-glycero-3-phosphocholine - 1،2-dioleoyl-sn-glycero-3-phosphocholineBSA - BSAPOPs - آلایندههای آلی دیرپاbovine serum albumin - آلبومین سرم گاوendothelin-1 - اندوتلین-1Cell-free - بدون سلولRoom temperature - دمای اتاقDodecylphosphocholine - دودهیل فسفو هولینdithiothreitol - دیتیوتریتولTCEP - ساکتcolumn volume - ستون حجمLiposome - لیپوزوم هاreaction mixture - مخلوط واکنشmembrane scaffold protein - پروتئین داربست غشاییSize exclusion chromatography - کروماتوگرافی اندازهای طردیImmobilized metal-ion affinity chromatography - کروماتوگرافی وابسته به فلز فلز یونیglutathione (oxidized) - گلوتاتیون (اکسید شده)Glutathione (reduced) - گلوتاتیون (کاهش یافته)
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Cell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the synthetic expression environment. We demonstrate the systematic quality optimization of the cell-free synthesized human endothelin B receptor by a combined approach of lipid screening, redox optimization, and molecular engineering. Key parameters for receptor folding are the implementation of nanodiscs, the selection of suitable lipid environments for co-translational solubilization, as well as providing an optimized redox system for essential disulfide bridge formation. In addition, enrichment with chaperones as well as receptor engineering by thermostabilization further supported the folding into ligand binding conformation. In summary, we provide evidence that the initial co-translational folding process rather than long-term stability of the receptor is limiting. The folding efficiency could be improved by more than 103-fold and under optimized conditions, up to 1.6â¯nmol or â¼100â¯Âµg of ligand binding competent receptor could be produced per mL of reaction mixture in a timescale of less than 24â¯h. The identified parameters affect rather common characteristics of G-protein receptors and are thus likely to improve the folding of similar targets as well. The optimized process provides full-length receptors embedded in defined membrane environments and in quantities and quality sufficient for throughput screening applications.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 147, 1 September 2018, Pages 73-83
Journal: Methods - Volume 147, 1 September 2018, Pages 73-83
نویسندگان
Ralf-Bernhardt Rues, Fang Dong, Volker Dötsch, Frank Bernhard,