Cloning, expression and characterization of the NERF-1a, ETS-domain protein.
Jaskulski, L.; Renard, G.; Werlang, I.C.R.; Batista, E.; Basso, L.A.; Santos, D.S.
Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas , Pontifícia Universidade Católica do Rio Grande do Sul/TECNOPUC - Porto Alegre Brazil.
The members of the Ets family share a highly conserved 80- to 90-amino-acid DNA binding domain, the Ets domain. This domain is sufficient to interact specifically with DNA sequences. Since the DNA binding domain is conserved, binding sites for Ets factors are all very similar, with a core binding motif, A/GGAA/T, and slight differences in flanking nucleotides for different Ets factors. Outside the DNA binding domain very little homology is common to all members of the Ets family. Ets related proteins can be grouped into subclasses based on additional homologous domains unique for particular members of the Ets family (Janknecht, R., and A. Nordheim, 1993, Biochim. Biophys. Acta 1155:346-356; Wang, C. Y., et al., Mol. Cell. Biol 14:1153-1159). NERF-1a is an isoform of a new Ets-transcription factor family member, as well as NERF/ELF-2, NER-2, NERF-1b. Both NERF-2 and NERF-1a could affect AML1 activity, whereas NERF-2 enhanced AML1-mediated transactivation of the B cell-specific blk promoter, NERF-1a drastically repressed AML1-mediated transactivation. The AML1 genes are the most frequent target for chromosomal translocation in human leukemias (Cho JY et al., 2004, J. Biol. Chem.). The work here presented represents a step towards a research project that aims at understanding the molecular mechanisms of DNA recognition by members of the Ets-domain family of eukaryotic transcription factors. Here we describe construction of DNA coding sequence of the Ets domain of NERF-1a. This construction was based on a technique in which overlapping oligonucleotides are PCR-extended by a DNA polymerase (patent deposited on INPI - 016050005891). The synthetic gene codifies for a 99 amino-acid protein that was cloned into pCR-Blunt vector and subcloned into pET 23a(+) expression vector. Different strains of Escherichia coli are being tested to overexpress the Ets domain of NERF-1a. Purification of the recombinant protein to homogeneity will provide enough material for crystallization trials and characterization of its three-dimensional structure. The biochemical and structural data will be used to classify the DNA binding activities aiming at building a DNA binding domain knowledge database to develop new analysis software and techniques for efficient and effective interpretation of experimental protein-DNA interaction data.
Supported by CNPq.
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