Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth.
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 alpha-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.
Cabos-Siguier
Béatrice
B
Group Interactions acides nucléiques/protéines comme cibles pharmacologiques, Université de Toulouse UPS/CNRS-IPBS (Institut de Pharmacologie et Biologie Structurale), Toulouse, France.
Steunou
Anne-Lise
AL
Joseph
Gérard
G
Alazard
Robert
R
Ducoux-Petit
Manuelle
M
Nieto
Laurence
L
Monsarrat
Bernard
B
Erard
Monique
M
Clottes
Eric
E
D000595:Amino Acid Sequence; D001686:Biological Phenomena; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008545:Melanoma / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050809:POU Domain Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D050809:POU Domain Factors; D010446:Peptide Fragments; C085076:transcription factor Brn-2; D014357:Trypsin