کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2022080 1069277 2006 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1
چکیده انگلیسی
Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN−wt1 and 6HIS-ZN+wt1, corresponding to two alternative splice variants of the C-terminal regions of human WT1 (−KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected ββα content for the refolded proteins, with a notable degradation of the α-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5′-GCG-TGG-GCG-3′ confirmed that 6HIS-ZN−wt1 has higher DNA binding affinity than 6HIS-ZN+wt1.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 46, Issue 2, April 2006, Pages 379-389
نویسندگان
, ,