Protein Expression and Purification of L. enzymogenes RpfB1 and RpfB2

Protein expression and purification were performed as described previously (35 (link)). To clone the L. enzymogenes rpfB1 and rpfB2 genes, genomic DNA extracted from L. enzymogenes was used for PCR amplification using Pfu DNA polymerase, and the primers are listed in Table 3. The PCR products were inserted into pET-28b (+) to produce the plasmids pET-rpfB1 and pET-rpfB1. The L. enzymogenes rpfB1 and rpfB2 genes were verified by nucleotide sequencing by Genscript (Nanjing, Jiangsu, China). rpfB1 and rpfB2 with a vector-encoded His₆-tagged N terminus were expressed in E. coli BL21(DE3) and purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). The protein purity was monitored by SDS-PAGE and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry.