کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843548 | 1069274 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site
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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
Histidine (His) tags are one of the most popular fusion tags for the isolation of proteins via metal affinity chromatography. The fusion tag is routinely left attached to the protein when carrying out experiments, with the assumption that the addition has no effect on structure or function. In the present study, we have prepared four proteins of the gene regulatory protein AreA from Aspergillus nidulans for crystallization experiments: a 91-amino acid peptide encompassing the minimal DNA-binding region, both with and without the His-tag (HZFB and ZFB, respectively), and a 155-amino acid protein previously proposed to be the entire DNA-binding domain for AreA, both with and without the His-tag (HG1b and G1b, respectively). To test the integrity of the four AreA proteins, urea denaturation experiments and DNA-binding studies were performed using fluorescence spectroscopy. The DNA-binding data showed similar dissociation constants for all proteins, with Kd values in the nanomolar range. The urea denaturation data, however, clearly indicated that the HZFB protein exhibited a completely different denaturation profile when compared to the ZFB, HG1b, and G1b proteins. The HZFB protein showed a profile indicative of the presence of an altered conformation around the sole tryptophan, whereas the other proteins showed a transition point between 3 and 4Â M urea concentration. These data show that, although function was not altered for any of the proteins studied, the structure of one of the His-tagged proteins was different from the native form of that protein.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 39, Issue 2, February 2005, Pages 152-159
Journal: Protein Expression and Purification - Volume 39, Issue 2, February 2005, Pages 152-159
نویسندگان
Alan Chant, Christina M. Kraemer-Pecore, Richard Watkin, Geoff G. Kneale,