A gene encoding the RC maquette was purchased from DNA2.0 in a pJexpress414 vector, and the DH maquette gene was purchased from GenScript in a pET-3a vector. Both genes included an N-terminal His6 tag followed by a TEV protease cleavage sequence. Invitrogen supplied mutagenesis primers, and mutant plasmids were PCR amplified with AccuPrime™ Pfx SuperMix (Invitrogen). Amino acid sequences are given in Supplementary Table S1 .
Plasmids were transformed into BL21-(DE3) competent cells (New England Biolabs), and proteins were expressed and purified as previously described (Ennist et al., 2022 (link)). Where noted, an additional purification step of high performance liquid chromatography (HPLC) was executed on a Waters reverse-phase HPLC system using a C4 or C18 HPLC column (Grace Davison Discovery Sciences). HPLC-pure samples were lyophilized and resuspended in >6.5 M GdnHCl, refolded by dilution, and purified by size exclusion chromatography (SEC) with Superdex 75 prep grade gel filtration medium (GE Healthcare Life Sciences).
Plasmids were transformed into BL21-(DE3) competent cells (New England Biolabs), and proteins were expressed and purified as previously described (Ennist et al., 2022 (link)). Where noted, an additional purification step of high performance liquid chromatography (HPLC) was executed on a Waters reverse-phase HPLC system using a C4 or C18 HPLC column (Grace Davison Discovery Sciences). HPLC-pure samples were lyophilized and resuspended in >6.5 M GdnHCl, refolded by dilution, and purified by size exclusion chromatography (SEC) with Superdex 75 prep grade gel filtration medium (GE Healthcare Life Sciences).
Free full text: Click here
