INVESTIGADORES
NERLI Bibiana Beatriz
artículos
Título:
Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization
Autor/es:
CHALLER, EMILSE; LISA, MARÍA NATALIA; NERLI, BIBIANA; CALCATERRA, NORA; ARMAS, PABLO
Revista:
PROTEIN EXPRESSION AND PURIFICATION
Editorial:
ACADEMIC PRESS INC ELSEVIER SCIENCE
Referencias:
Lugar: Amsterdam; Año: 2014 vol. 93 p. 23 - 31
ISSN:
1046-5928
Resumen:
Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advancesmade concerningCNBPbiological roles, a full knowledge about the structure?function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here,we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan,which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and b-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBPstructural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding.