کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360464 | 1542340 | 2014 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6Â ÃÂ His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6Â ÃÂ His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40Â mg purified and active protein was obtained from 1Â L of cell culture. This recombinant 6Â ÃÂ His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca2+-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 101, September 2014, Pages 28-36
Journal: Protein Expression and Purification - Volume 101, September 2014, Pages 28-36
نویسندگان
Angelo Verlotta, Daniela Trono,