کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021339 1069241 2009 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth
چکیده انگلیسی

This report describes the first purification procedure of the human full-length N Oct-3 protein in amounts suitable for structural studies and proteomic investigations. N Oct-3 is a transcription factor member of the POU protein family. It possesses a large N-terminal transactivation domain and a DNA-binding domain (DBD) which is composed of two subdomains, POUs and POUh, which are joined by a linker peptide. N Oct-3 is a master gene for central nervous system development but also for melanoma progression. Previous structural studies have all been performed using N Oct-3 DBD only. In this study, the full-length N Oct-3 protein was bacterially expressed and purified to homogeneity. The purified protein gave a single band at approximately 53 kDa on SDS–PAGE, while cDNA sequence analysis revealed a calculated molecular mass of 47 kDa confirmed by mass spectroscopy. Size-exclusion chromatography experiments indicated that in solution, full-length N Oct-3 was a monomer. Circular dichroïsm and intrinsic tryptophan fluorescence showed that full-length N Oct-3 was folded, with a significant α-helix content probably located in its DBD. Comparison with the purified N Oct-3 DBD demonstrated that, at least in vitro, the affinity of the protein for its DNA targets was similar. This suggests that the transactivation domain of N Oct-3 was not involved in N Oct-3 DNA interaction.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 64, Issue 1, March 2009, Pages 39–46
نویسندگان
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