Purification and characterization of the cellular nucleic acid-binding protein CNBP

CHALLIER, E.; LISA, MN; CALCATERRA, N.B.; ARMAS, P

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

SAB

The Cellular Nucleic Acid-Binding CNBP is a single-stranded RNA and DNA binding protein that recognizes G-rich sequences1. CNBP shows nucleic acid chaperone activity1,2 and has an important role in vertebrate craniofacial structures embryogenesis3. CNBP primary structure consists of 7 Zn knuckles of the type CCHC and a RGG box between the first and second Zn knuckles, being the first Zn knuckle and the RGG box essential for its biochemical activity4. In this work we have cloned zebrafish (Daniorerio) CNBP cDNA as a Thioredoxine translational fusion, separated by an (His)6 linker and the specific TEV protease site. The CNBP purification has been achieved by two affinity chromatographies in Ni+2 columns with an intermediate TEV digestion step and a last purification by molecular exclusion chromatography. By means of electronic absorption spectroscopy we determined the presence of nucleic acids that co-purified with CNBP.. We obtained a unique truncated CNBP form by autoproteolysis of the complete purified form, which was analyzed by N-terminal sequencing for determining the precise proteolysis site. When co-purified with nucleic acids, CNBP was more stable to proteolysis and precipitation, suggesting a more rigid CNBP structure in the complex with nucleic acids. As CNBP was detected as a complete and truncated protein when obtained from embryo extracts, and the proteolysis may eliminate the N-terminal motifs relevant for its biochemical function, it is tempting to speculate about a regulatory function of the proteolysis site