STRUCTURAL CHARACTERIZATION OF THE RNA BINDING DOMAIN OF HUMAN STEM LOOP BINDING PROTEIN

Maruthi Kashyap, Neel Sarovar Bhavesh

Abstract


A gene encoding the RNA binding domain (RBD) of human stem loop binding protein (SLBP) wascloned in pET 28a vector and over-expressed in E. coli codon plus cells. The over-expressed SLBP-RBD carried no tag and aggregated as inclusion bodies in the cell lysate. Inclusion bodies were semi-purified to >85% purityby establishing a method involving detergent washing and subsequently denatured in 8 M urea. Refolding ofthe denatured RBD was carried out by step dialysis in decreasing concentrations of urea and L-arginine. RefoldedSLBP-RBD was analyzed using size exclusion chromatography that revealed its monomeric nature and foldedstate. Uniformly 15N and 15N, 13Nlabeled SLBP-RBD was prepared at concentrations for solution NMR studies. Approximately, 60% of the sequence specific backbone resonance assignments have been achieved throughstandard triple resonance NMR experiments. Analyses of secondary chemical shifts reveal presence of a smallhelical secondary structural elements and large intrinsically disordered regions.
Keywords: Human SLBP refolding; Refolded RBD (r-RBD); Solution NMR; Backbone assignment


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