Site-Directed Mutagenesis of Helicobacter pylori CagL

Site-directed mutagenesis of CagL was performed using the pAD1 vector as DNA template [9] (link). As shown in Figure 1B, CagL of GC strains contain the amino acids Tyr-Glu-Ile-Gly-Lys (YEIGK) at position 58–62 of the protein, while strain 26695 has the amino acids Asn-Glu-Met-Gly-Glu (NEMGE) at this position. To generate a CagL 26695^YE mutant (Figure 1B, bottom), we performed PCR reactions with the following primers: 628F 5′-GGTAAAGAAGATGCTCTAAACATC and 628R 5′-GATTTCATAATTAGCACTAGGGCTAG to open and amplify the entire construct. For amplification, Phusion^® High-Fidelity DNA Polymerase (NEB, Ipswich, USA) was used, followed by PCR purification (MinElute PCR Purification Kit, Qiagen, Hilden, Germany), digestion with DpnI (Promega, Madison, USA), and ligation using T4 DNA Ligase (Promega). Re-sequencing and Western blotting of E. coli or H. pylori lysates, respectively, verified the appropriate expression of CagL mutant variants from the resulting plasmids.