کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1200328 1493579 2014 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Sequential tentacle grafting and charge modification for enhancing charge density of mono-sized beads for facilitated protein refolding and purification from inclusion bodies
ترجمه فارسی عنوان
پیوند قاعدگی به طور متوالی و تغییر شار برای افزایش تراکم شارژ مهره های منحصر به فرد برای تسهیل پروتئین و تصفیه از بدن
کلمات کلیدی
مهره های چوبه دار، تغییر، سطح شارژ منفی، بازخورد پروتئین، پاکسازی، میکروسفور یکنواخت
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی


• Sequential grafting and charge modification enhanced sulfonate density to 793 μmol/g.
• Enhanced EGFP refolding and purification were realized with the highly charged beads.
• 52% of contaminant proteins was removed by adsorption to the charged beads.
• Reusability of the charged beads was demonstrated in recycled protein refolding.

We have previously found that addition of like-charged media in a refolding solution can greatly enhance the refolding of pure proteins by suppressing protein aggregation. Herein, negatively charged mono-sized microspheres with sulfonic groups were fabricated to explore the facilitating effect of like-charged media on the refolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies (IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (pGMA) beads (2.4 μm). Namely, GMA was first grafted onto the beads by grafting polymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxy groups on the tentacles were converted into sulfonic groups by modification with sodium sulfite. By this fabrication strategy, the charge density of the beads reached 793 μmol/g, about 2.8 times higher than that modified without prior grafting of the pGMA beads (285 μmol/g). The negatively charged beads of different charge densities were used for facilitating the refolding of like-charged EGFP from IBs. The refolding yield as well as refolding rate increased with increasing charge density. The anti-aggregation effects of urea and like-charged microspheres were synergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to 52% removal of positively charged contaminant proteins in the refolded solution. Finally, reusability of the tentacle beads was demonstrated by repetitive EGFP refolding and recovery cycles.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1347, 20 June 2014, Pages 49–55
نویسندگان
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