Expressão, purificação e caracterização estrutural dos fatores de transcrição bZIP SCF12 e SCF5 de cana-de-açucar / Expression, purification and structural characterization of the sugarcane bZIP transcription factors SCF12 and SCF5

AUTOR(ES)

Eduardo Kiyota

DATA DE PUBLICAÇÃO

2008

RESUMO

The bZIP transcription factors are present in eukaryotic organisms and are involved in the regulation of gene expression and many intracellular processes. These factors bind specific DNA sequences and are able to recognize regulatory sequences of a gene promoter. The bZIPs are characterized by a conserved region rich in basic amino acid residues as well as by having the leucine zipper region, which possess a sequence of hydrophobic residues where there are leucines every seventh amino acids. Structural studies have shown that bZIP-folding is alpha-helical and these proteins are capable of dimmer formation via coiled-coil arrangement. In this work, the basic region and the leucine zipper of two sugarcane bZIPs, SCF12 and SCF5, belonged to two different bZIP-families were cloned, expressed and purified for structural studies. The corresponding SCF12 DNA was cloned into pET28a expression vector and the protein was produced in E. coli BL21 (DE3) pRil cells. SCF12 protein was purified by affinity chromatography (IMAC) and had its secondary structure characterized by CD. SCF5, cloned into pET3c and expressed in E. coli BL21 (DE3) pLysS was purified by cation exchange chromatography. Crystals of a complex formed by SCF5 protein and a 24-base-pair DNA sequence were obtained but unfortunately with quality insufficient for crystallographic structure determination. However, it was possible to obtain a model of the analyzed complex applying Small Angle X-ray Scattering (SAXS) technique by protein homologous structure comparison.

ASSUNTO(S)

small-angle x-ray scattering raios x - espalhamento a baixo angulo fatores de transcrição bzip bzip transcription factors sugar-cane cristalografia de proteinas cana-de-açúcar protein crystallography

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