CMD1 recombinant protein expression and purification.

The ORF of CMD1 (Cre12.g553400.t2.1, Phytozome) was cloned into modified pET28a (pPEI-His-SUMO supplied by Yanhui Xu)¹⁸ and the construct was transformed into E. coli strain BL21 (DE3). The CMD1 mutants were constructed in the same vector. The bacterial cells grown to an absorbance of 0.8 at OD600 were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 °C for 16 h. Tagged CMD1 protein was bound to Ni-NTA beads (Qiagen) and cleaved off from the His-SUMO tag by overnight incubation with His-tagged Ulp1 protease at 4 °C. The collected CMD1 protein was further purified using a Resource Q anion exchange column (GE Healthcare) with a linear gradient of buffer A (20 mM Tris-HCl, pH 8.5)/buffer B (20 mM Tris-HCl, pH 8.5, 1 M NaCl) from 100/0 to 50/50, and a Superdex 200 10/300 GL gel filtration column (GE Healthcare) in buffer (20 mM HEPES pH 7.0, 100 mM NaCl). The protein was concentrated to 10 μg/μl using Ultracel-10K centrifugal filters (Millipore).