کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020160 1542313 2016 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease
ترجمه فارسی عنوان
یک روش ساده برای تصفیه پروتئین نوترکیب با استفاده از پروتئاز ویروس اچ ویروس پنهان شده با پپتید با برچسب خودپرداز
کلمات کلیدی
تنباکو اچ ویروس پروتئاز، پپتید خودمراقبتی، مخلوط پروتئین فعال، تصفیه پروتئین
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
چکیده انگلیسی


• We constructed an N-terminal self-assembling peptide ELK16 fusion expression vector.
• We expressed ELK16-tagged tobacco etch virus protease as active inclusion bodies.
• We demonstrated the high activity of ELK16-tagged protease for cleavage of His-tag.
• We showed that the ELK16-tagged protease could be removed from the digest by centrifugation.
• We purified recombinant bovine interferon-γ to high purity using the ELK16-tagged protease.

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 104 units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 103 units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 128, December 2016, Pages 86–92
نویسندگان
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