کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10537615 | 962794 | 2005 | 12 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Complete refolding of bovine β-lactoglobulin requires disulfide bond formation under strict conditions
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کلمات کلیدی
PBSGdnHClβ-LgGSSGGSHmAb2-(N-morpholino) ethanesulfonic acid - 2- (N-مورفولینو) اتان سولفونیک اسیدβ-Lactoglobulin - β-لاکتوگلوبولینMonoclonal antibody - آنتی بادی مونوکلونالRefolding - بازپرداختDisulfide bond formation - تشکیل پیوند دی سولفیدEquilibrium constant - تعادل ثابتDissociation constant - حد تفکیکoxidized form of glutathione - شکل اکسید شده گلوتاتیونPhosphate-buffered saline - محلول نمک فسفات با خاصیت بافریMeS - مسKAS - چیreduced form of glutathione - کاهش شکل گلوتاتیونGuanidine hydrochloride - گوانیدین هیدروکلراید
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله

چکیده انگلیسی
β-Lactoglobulin (β-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414-22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed β-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured β-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured β-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1752, Issue 2, 25 September 2005, Pages 154-165
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1752, Issue 2, 25 September 2005, Pages 154-165
نویسندگان
Makoto Hattori, Kazuhiko Hiramatsu, Takashi Kurata, Mika Nishiura, Koji Takahashi, Akio Ametani, Shuichi Kaminogawa,