Bamhi and hindiii restriction enzymes

Escherichia (E.) coli DH5α cells were grown in LB broth (Sigma Chemical Co., St. Louis, MO, USA) at 37 °C with shaking. CHO cells, pcDNA™3.1(+) vector, BamHI and HindIII restriction enzymes, and kits for plasmid extraction were purchased from Thermo Fisher Scientific (USA).

Peptides were grafted onto the CTPR2 construct reported by Grove et al. (2010 (link)) with the C-terminal NN residues mutated to RS as reported by Phillips et al. (2012 (link)). The grafted peptide is flanked by the DPNN sequence at its N-terminus and the DPNS sequence at its C-terminus. This sequence was chosen as DPNN is the ‘native’ inter-repeat loop sequence of the CTPR and the N→S mutation was made to improve protein solubility (Table I).
The CTPR constructs were cloned using gBlock oligos (Integrated DNA technologies) into the multiple cloning site of the pRSET B vector using restriction digestion-ligation cloning with BamHI and HindIII restriction enzymes (ThermoFisher Scientific, Waltham, MA) and Quick Stick ligase (Bioline). The Keap1 Kelch domain construct (residues 321–624) was a kind donation from Alex Bullock (Structural Genomics Consortium, Oxford) with an N-terminal His-tag and a TEV cleavage site in a pNIC28-BSA4 vector.