title: Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.
abstract: 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 advances made concerning CNBP biological 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 β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural 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.
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CCT-Rosario, Ocampo y Esmeralda, S2000FHQ Rosario, Argentina.
authors.forename: Emilse
authors.initials: E
authors.lastname: Challier
authors.forename: María-Natalia
authors.initials: MN
authors.lastname: Lisa
authors.forename: Bibiana B
authors.initials: BB
authors.lastname: Nerli
authors.forename: Nora B
authors.initials: NB
authors.lastname: Calcaterra
authors.forename: Pablo
authors.initials: P
authors.lastname: Armas
keywords: Cellular nucleic acid binding protein; Intrinsic fluorescence quenching; Intrinsically unstructured protein; Nucleic acid binding; Proteolysis assay; Tag-free; Zinc knuckle