Cloning and Purification of RpfG Protein

Protein expression and purification were performed as follows^{42 (link)}. To clone the rpfG gene, genomic DNA extracted from L. enzymogenes was used for PCR amplification using Pfu DNA polymerase, and the primers are listed in Supplementary Table 2. The PCR products were inserted into pMAl-p2x to produce the plasmids pMAl-rpfG. The rpfG gene was verified by nucleotide sequencing by Genscript (Nanjing, Jiangsu, China). Le rpfG and rpfG site-directed mutants with a vector-encoded maltose binding protein in the N-terminus were expressed in E. coli BL21 (DE3) and purified with Dextrin Sepharose High Performance (Qiagen, Chatsworth, CA, USA) using an affinity column (Qiagen). The protein purity was monitored by SDS-PAGE. His₆-tagged protein expression and purification were performed as described previously^{41 (link)–43 (link)}.