کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7617773 | 1494083 | 2014 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves
ترجمه فارسی عنوان
یک فرآیند پس از آن باعث انسداد کارآمد پروتئین آمفیفیفی نوترکیب شده از برگ های توتون و تنباکو می شود
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 960, 1 June 2014, Pages 34-42
Journal: Journal of Chromatography B - Volume 960, 1 June 2014, Pages 34-42
نویسندگان
Elisa Gecchele, Stefan Schillberg, Matilde Merlin, Mario Pezzotti, Linda Avesani,