Generation and Purification of Cmr2 and Palm Mutants

Cmr2 mutants HD_m (H13A, D14A) and Palm_m (D673A, D674A) were generated by PCR mutagenesis (primers in Supplemental Table S2). Expression and purification of both recombinant wild type and mutants, including Cmr4-D26N (Ramia et al. 2014a (link)), was performed as described (Hale et al. 2009 (link), 2014 (link)) with the following modifications: Cells were lysed, and protein was purified in buffer containing 40 mM Tris-Cl (pH 7.5), 500 mM NaCl, and 0.2 mM PMSF. Purified proteins were dialyzed into 40 mM Tris-Cl (pH 7.5) and 500 mM NaCl and quantified by Qubit assay (Life Technologies).