کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843341 | 1069219 | 2010 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells
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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
Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7Â days of induction the expression levels of the full-length and truncated versions were 5Â mg/l and 2Â mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 72, Issue 1, July 2010, Pages 87-94
Journal: Protein Expression and Purification - Volume 72, Issue 1, July 2010, Pages 87-94
نویسندگان
Morten G. Rasch, Ida K. Lund, Martin Illemann, Gunilla Høyer-Hansen, Henrik Gårdsvoll,