کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360266 | 1542333 | 2015 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Improved Production and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor from E. coli under Optimized Downstream Processes
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coli BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4 g/L) and purified rhG-CSF (136 mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8 M urea at pH 9.5. Refolding efficiency studies showed maximum refolding ⩾86% and ⩾84% at 20 °C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (⩾95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with ⩾98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8 kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 108, April 2015, Pages 62-72
Journal: Protein Expression and Purification - Volume 108, April 2015, Pages 62-72
نویسندگان
Sandeep Vemula, Rahul Thunuguntla, Akshay Dedaniya, Sujana Kokkiligadda, Chaitanya Palle, Srinivasa Reddy Ronda,