کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
3407294 | 1593497 | 2010 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
High yield expression and purification of HIV-1 Tat1â72 for structural studies
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کلمات کلیدی
DSSIPTGDTTβMEHexim1HSQCCDK92,2-dimethyl-2-silapentane-5-sulfonate - 2،2-dimethyl-2-silapentane-5-sulfonateβ-mercaptoethanol - β-merkaptoethanolisopropyl-β-d-thiogalactopyranoside - ایزوپروپیل-ب-دی-تیوگالکتوپیرانوزیدHAD - داشته استHIV-associated dementia - دمانس مرتبط با HIVdithiothreitol - دیتیوتریتولMatrix assisted laser desorption/ionization time-of-flight mass spectrometry - ماتریکس از اسپکترومتر جرمی زمان رسیدن به یخ زدن لیزر استفاده کردMALDI-TOF-MS - مالدی TOF-MSHive - کندوCyclin-dependent kinase 9 - کیناز وابسته به سیکلین 9heteronuclear single quantum coherence - یکپارچگی کوانتومی تک هسته ای
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ویروس شناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of 6ÃHistidine-tagged and isotopically enriched (in N15 and C13) recombinant HIV-1 Tat1â72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 164, Issues 1â2, March 2010, Pages 35-42
Journal: Journal of Virological Methods - Volume 164, Issues 1â2, March 2010, Pages 35-42
نویسندگان
Shaheen Shojania, Gillian D. Henry, Vincent C. Chen, Thach N. Vo, Hélène Perreault, Joe D. O'Neil,