کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843792 | 1069293 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Cloning, expression, characterization, and crystallization of a glutaminyl cyclase from human bone marrow: A single zinc metalloenzyme
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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
Glutaminyl cyclase (QC) catalyzes the N-terminal pyroglutamate formation of numerous hormones and peptides from their glutaminyl precursor. Pyroglutamate is a posttranslational or cotranslational modification important in many physiological and pathological processes. Here, we present the cloning of a QC cDNA from human bone marrow cDNA library. The protein was expressed in Escherichia coli system with the yields higher than â¼10Â mg/L bacterial culture, using a thioredoxin-tagged expression vector with several modifications. Based on high histidine content (â¼5%) of the protein, a convenient purification step by Ni-affinity chromatography was designed, leading to near homogeneity of the purified human QC. The identity of the recombinant human QC was confirmed by mass spectrometry and circular dichroism spectroscopy. The enzyme was active on both synthetic and physiological substrates, and the activity could be inhibited by several imidazole, triazole, and tetrazole derivatives. An atomic absorption analysis demonstrated that human QC contains one zinc ion per protein molecule. We also obtained the human QC crystals, which belong to cubic, tetragonal, and rhombohedral forms. Our works are useful to acquire new insights into human and animal QCs, particularly for future structural analysis and inhibitor designs.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 43, Issue 1, September 2005, Pages 65-72
Journal: Protein Expression and Purification - Volume 43, Issue 1, September 2005, Pages 65-72
نویسندگان
Kai-Fa Huang, Yi-Liang Liu, Andrew H.-J. Wang,