کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
6491042 43390 2015 26 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Strategy for linker selection to enhance refolding and bioactivity of VAS-TRAIL fusion protein based on inclusion body conformation and activity
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Strategy for linker selection to enhance refolding and bioactivity of VAS-TRAIL fusion protein based on inclusion body conformation and activity
چکیده انگلیسی
A bifunctional fusion protein, VAS-TRAIL, was designed for superior therapeutic efficacy by combining anti-angiogenesis activity with tumor-selective apoptosis activity. The protein was expressed as inclusion body (IB) in Escherichia coli. To enhance refolding yield and bioactivity, four fusions were constructed with different linkers (no linker, flexible linker, rigid linker, and helix-forming linker). A novel linker selection strategy based on IB conformational quality and activity was applied to predict the suitable linker. The conformational quality and activity of VAS-TRAIL IBs were analyzed by ATR-FTIR and cytotoxicity assay, respectively. Results demonstrated that aggregated VRT (fusion with rigid linker) contained the highest native-like β structure content and retained part of the expected activity, namely, cytotoxicity activity on tumor cells. This finding suggested that the rigid linker was the most suitable candidate. Further results of in vitro refolding and subsequent circular dichroism and activity assay of four refolded fusions were significantly correlated with the predictions. Refolding of VRT yielded more soluble proteins containing the expected secondary structure and the highest bioactivity compared with that of other fusions. Our research may offer an efficient method for the high-throughput design of aggregated-prone therapeutic fusion protein.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 209, 10 September 2015, Pages 16-22
نویسندگان
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