Recombinant Protein Production and Purification

All recombinant proteins were produced in Escherichia coli strain KRX (Promega) using isopropyl β-D-1-thiogalactopyranoside and rhamnose for induction of protein expression. The MRPP1/2 complex was produced essentially as previously described (19 (link)) and stored in 20 mM Tris pH 7.6, 200 mM NaCl, 10%(v/v) glycerol and 2 mM Tris(2-carboxyethyl)phosphine (TCEP). MRPP3 was purified as previously reported (19 (link)) and stored in 20 mM Na-HEPES pH 7.5, 300 mM NaCl, 10%(v/v) glycerol and 0.5 mM TCEP. ELAC2 was purified using nickel-affinity, Q anion exchange and size exclusion chromatography in a final buffer of 50 mM Tris pH 8.0, 150 mM NaCl, 10%(v/v) glycerol and 5 mM TCEP. The N-terminal His₆-tag was removed by TEV protease treatment. The CCA-adding enzyme was purified using nickel-affinity, heparin affinity and size exclusion chromatography in a final buffer of 20 mM Na-HEPES pH 7.6, 150 mM NaCl, 5%(v/v) glycerol and 2 mM TCEP. All proteins were concentrated by ultrafiltration and then flash cooled in liquid nitrogen and stored at −80°C.