Rh Bri2 BRICHOS R221E and Bri2 BRICHOS-AU1 preparation
For generating rh Bri2 BRICHOS R221E the primers 5′-CACCTGGGTTTCTTTATTTATGAACTGTGTCATGACAAGGAAAC-3′ and 5′- GTTTCCTTGTCATGACACAGTTCATAAATAAAGAAACCCAGGTG-3′ were synthesized. With the wild-type NT*-Bri2 BRICHOS (corresponding to the solubility tag NT* followed by Bri2 residues 113–23126,41) plasmid as polymerase chain reaction (PCR) template Bri2 BRICHOS R221E (Addgene ID: 138134) was obtained with QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, US), and the DNA sequence was confirmed (GATC Bioteq, Germany). As described previously26,41, the proteins were expressed in SHuffle T7 competent Escherichia coli cells. Briefly, the cells were incubated at 30 °C in LB medium with 15 µg mL−1 kanamycin until an OD600 nm ~0.9. For overnight expression, the temperature was lowered to 20 °C, and 0.5 mM…
