کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7559029 | 1491382 | 2014 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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![عکس صفحه اول مقاله: Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures](/preview/png/7559029.png)
چکیده انگلیسی
Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8Â M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2Â M N-ethylurea and 8Â M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pKa values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0-3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 465, 15 November 2014, Pages 90-95
Journal: Analytical Biochemistry - Volume 465, 15 November 2014, Pages 90-95
نویسندگان
David Gervais, Darryl King,