XXXV Reunião Anual da SBBqResumoID:8477


Characterization of the Ets domain of Human PE1: Gene Construction, Cloning and Expression.


Gianniotis, E.; 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, Pontificia Universidade Católica do Rio Grande do Sul / TECNOPUC. Avenida Ipiranga, 6681, Prédio 92A – Porto Alegre-RS, CEP 90619-900 – Brazil.


The Ets gene family encodes eukaryotic transcription factors that bind specific DNA sequences through the highly conserved, 85-residue Ets domain. This region forms the winged helix-turn-helix DNA binding domain composed of three alpha helices and a four-stranded beta sheet that recognizes a core GGAA/T sequence (Donaldson LW et al., 1994, Biochemistry, 33, 13509-13516). The Ets factors are known to act as positive or negative regulators of the expression of genes that are involved in various biological processes, including those that control cellular proliferation, differentiation, hematopoiesis, apoptosis, metastasis, tissue remodeling, angiogenesis and transformation. Ets repressors are candidates for proteins that might differentially regulate programs of proliferation and differentiation and the Ets repressor PE1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation (Sawka-Verhelle et al., 2004, The Journal of Biological Chemistry, 23, 17772-17784). PE1 can be found in a large variety of cancer cell lines and is highly expressed in hematopoietic cells and in the primitive hematopoietic cells (Klemsz et al., 1994, Genomics 20:291-294). The work here presented is part of an ongoing project that focuses on the understanding of molecular mechanisms of DNA recognition by members of the Ets-domain family of eukaryotic transcription factors. 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. Accordingly, the gene sequence that codes for the 101 amino-acid Ets domain of PE1 was synthetically constructed by the PCR extension of overlapping oligonucleotides by a DNA polymerase (patent deposited on INPI - 016050005891). The PCR amplified DNA fragment was cloned into pCR-Blunt vector and subcloned into pET 23a(+) expression vector using NdeI and HindIII restriction enzymes. The latter construction was sequenced and protein expression assays using different Escherichia coli strains are currently underway. These results will pave the way for protein purification and crystallization trials aiming at its three-dimensional structure determination.

Supported by CNPq.