Polyhistidine

The MeCP2 variants (corresponding to isoform E2) were expressed using pET30b plasmid as expression vector for BL21 (DE3) Star E. coli strain. The sequences contained an N-terminal polyhistidine-tag for purification by immobilized metal ion affinity chromatography (IMAC). Cultures were grown in 150 ml of LB/kanamycin (50 μg/ml) media at 37°C overnight. Then, 4 l of LB/kanamycin (25 μg/ml) were inoculated (1:100 dilution) and incubated under the same conditions until reaching an OD (at a wavelength of 600 nm) of 0.6. Protein expression was induced with 1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG) at 18°C overnight. Cells were ruptured by sonication in ice and benzonase (Merck-Millipore, Madrid, Spain) was added (20 U/ml) to degrade nucleic acids.
Proteins were purified in a HiTrap TALON column (GE-Healthcare Life Sciences, Barcelona, Spain) with two washing steps: buffer sodium phosphate 50 mM, pH 7, NaCl 300 mM, and in buffer sodium phosphate 50 mM, pH 7, NaCl 800 mM (to remove potential DNA contamination from the protein), before an imidazole 10–150 mM elution gradient. Purity was checked by SDS-PAGE. The polyhistidine-tag was removed by GST-tagged PreScission Protease cleavage in buffer Tris–HCl 50 mM, pH 7.5, NaCl 150 mM, at 4 °C for 4 h. The final step consisted of a combination of two affinity chromatographic steps: removal of the polyhistidine-tag, or the unprocessed protein, using a HiTrap TALON column (GE-Healthcare Life Sciences, Barcelona, Spain) and removal of the GST-tagged PreScission Protease using a GST TALON column (GE-Healthcare Life Sciences, Barcelona, Spain). Purity and homogeneity were checked by SDS-PAGE. The proteins were stored in buffer Tris 50 mM, pH 7.0 at −80°C. Potential DNA contamination was always assessed by the ratio of UV absorption at 260 nm versus absorption at 280 nm. Extinction coefficients at 280 nm of 11 460, 13 075 and 16 960 M⁻¹ cm⁻¹ were employed for wild-type/R133C MBD, full-length MeCP2, and R106W MBD, respectively.