کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843516 | 1069269 | 2005 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Two methods for large-scale purification of recombinant human choline acetyltransferase
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کلمات کلیدی
hexahistidine tag - برچسب hexahistidineKinetic analysis - تجزیه سینتیکisoelectric focusing - تمرکز ذره ای الکتریکیProtein purification - خالصسازی یا تخلیص پروتئینChitin-binding domain - دامنه اتصال چیتینMass spectrometry - طیف سنجی جرمیSpecific activity - فعالیت خاصgel filtration - فیلتر کردن ژلcholine acetyltransferase - کولین استیل ترانسفراز
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Two methods for large-scale purification of recombinant human choline acetyltransferase Two methods for large-scale purification of recombinant human choline acetyltransferase](/preview/png/10843516.png)
چکیده انگلیسی
Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 μmol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 40, Issue 1, March 2005, Pages 107-117
Journal: Protein Expression and Purification - Volume 40, Issue 1, March 2005, Pages 107-117
نویسندگان
Ae-Ri Kim, Amanda Doherty-Kirby, Gilles Lajoie, R. Jane Rylett, Brian H. Shilton,