IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Set-up of a reproducible procedure to purify CNBP suitable for structural and biochemical studies.
Autor/es:
CHALLIER, E.; LISA, M. N.; NERLI, B. B.; CALCATERRA, N. B.; ARMAS, P.
Lugar:
Potrero de los Funes
Reunión:
Congreso; XLVII Annual Meeting of the Argentine Society for Biochemestry and Molecular Biology Research.; 2011
Institución organizadora:
Society for Biochemestry and Molecular Biology Research.
Resumen:
Cellular nucleic acid binding protein (CNBP) is a conserved zincknuckle-containing single-stranded nucleic acid binding proteininvolved in human muscular diseases and craniofacialdevelopment. Recombinant CNBPs fused to different tags wereused for previous biochemical studies. However, tag-free CNBP isneeded to perform spectroscopic approaches. Here we describe apurification procedure that yielded 5 mg of soluble tag-freerecombinant CNBP per liter of LB. Electrophoretic mobility shiftassays and intrinsic fluorescence quenching experiments showedthat purified CNBP is biochemically active. Size exclusionchromatography revealed homodimeric and monomeric formscoexisting in solution independently of CNBP binding to its targets.CD spectra predicted that CNBP secondary structure is dominatedby ß-sheet, probably comprising zinc knuckles, and has highcontent of random-coil. CNBP zinc knuckles are implicated in itsstructure and functionality since Zn2+-depleted CNBP failed to bindsingle-stranded nucleic acids and was more sensitive to proteolysis.Moreover, CNBP was less sensitive to proteolysis in the presence ofsingle-stranded DNA or RNA, suggesting that CNBP gainsstructure upon binding to its targets. The availability of thepurification method presented here will be useful for furtherstructural and biochemical characterization to shed light on CNBPbiological function.