کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1974589 1060359 2009 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli
چکیده انگلیسی

Parvalbumins (PV) are small, acidic, water soluble and calcium-binding proteins generally present in muscular and nervous tissues. In the present study, we identified and characterized a cDNA clone encoding PV, named AplPV, from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. AplPV belongs to EF-hand proteins with six α-helices constituting three EF-hand domains. The deduced amino acid sequence of AplPV is 91% and 68% identical to the previously characterized PVs of Boa constrictor and Cyprinus carpio, respectively. The full-length cDNA was subcloned into the expression vector pGEX and transformed into Escherichia coli (E.coli) to produce recombinant protein. The bacterially expressed GST–AplPV fusion protein was highly expressed, and effectively purified by Glutathione–Sepharose affinity chromatography. A high concentration of thrombin protease specifically cleaved and removed the GST tag from fusion protein, and further purified by Benzamidine column for removal of thrombin protease. As a result, the 12 kDa AplPV recombinant protein alone was purified. To investigate the tissue-specific biological occurrence of AplPV, a polyclonal antibody (anti-AplPV-antibody) was raised against GST–AplPV fusion protein in rabbit. Western blot analysis revealed that immunoreactive bands were exhibited in both recombinant protein and samples of venom glands, but not in any crude venom. This specific occurrence indicates a specialized function of AplPV in snake venom glands.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology - Volume 153, Issue 3, July 2009, Pages 303–308
نویسندگان
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