Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth

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• 作者：Bé ; atrice Cabos-Siguier ; Anne-Lise Steunou ; Gé ; rard Joseph ; Robert Alazard ; Manuelle Ducoux-Petit ; Laurence Nieto ; Bernard Monsarrat ; Monique Erard ; Eric Clottes
• 关键词：POU proteins ; Circular dichroï ; sm ; Fluorescence ; Footprinting ; EMSA
• 刊名：Protein Expression and Purification
• 出版年：2009
• 出版时间：March 2009
• 年：2009
• 卷：64
• 期：1
• 页码：39-46
• 全文大小：687 K

文摘

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.