Purification and Characterization of HP1043 Protein

Recombinant N-terminal His-tagged HP1043 wild-type and mutant proteins were overexpressed in E. coli DH5α cells transformed with plasmid pTrc::1043 and its derivatives. For DNase I footprinting assays, HP1043 was affinity-purified as previously described [13 (link)] and dialyzed against two changes of 1× 1043 Footprinting Buffer (1× 1043 FPB: 10 mM Tris-HCl pH 7.5; 50 mM NaCl; 10 mM MgCl₂; 1 mM DTT; 0.01% Igepal CA-630; 10% glycerol). For EMSA assays, HP1043 was purified as in [34 (link)]) and dialyzed against the store buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 10% glycerol). Protein purity was assessed by SDS-PAGE and the concentration of protein preparations was estimated by Bradford colorimetric assay (BioRad, Hercules, CA, USA). HP1043 mutants were obtained by all-around PCR performed on plasmid pTrc::1043 with divergent primers listed in Table S1.