کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843672 | 1069287 | 2005 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in 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
Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 Ã
resolution with synchrotron radiation.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 44, Issue 2, December 2005, Pages 130-135
Journal: Protein Expression and Purification - Volume 44, Issue 2, December 2005, Pages 130-135
نویسندگان
Tetsuya Shimizu, Hiroyuki Shibata, Tomoyuki Araya, Toru Nakatsu, Kazuo Miyairi, Toshikatsu Okuno, Hiroaki Kato,