Abstract
Alpha‐fetoprotein (AFP) is one of the major serum proteins in the early life of mammals. We have previously identified a novel cis‐acting element designated as DAS at the 5′‐flanking region of the AFP gene and demonstrated that the DAS sequence can be specifically recognized by nuclear protein DAP‐II in AFP‐producing hepatoma cells and retinoic acid (RA)‐induced AFP‐producing F9 cells. In this study, we used DNA affinity chromatography to purify the DAP‐II proteins from the nuclear extracts (NE) of RA‐treated F9 cells. The purified DAP‐II complex mainly contained five proteins, with molecular weights of 45, 42, 32, 30, and 20 kDa, respectively. The identification of these proteins was determined by MALDI‐TOF mass spectrometric analysis and a database search. These proteins were found to belong to the AUF1 RNA‐binding protein family. Protein (30 kDa), one of five proteins in an isolated DAP‐II complex, was matched with amino acid sequence highly similar to muAUF1‐3. The expression of this protein is inducible by RA, and the pattern of the protein expression is the same as DAP‐II proteins in F9 cells after treatment with RA during differentiation. Our results suggest that the 30‐kDa protein is a novel isoform of AUF1 family and is the main component of the DAP‐II complex that binds to the DAS sequence. J. Cell. Biochem. 98: 1257–1270, 2006. © 2006 Wiley‐Liss, Inc.