FIG. 1.
Construction of mutagenized malE-papA plasmids. papA DNA fragments were synthesized by PCR amplification under random mutagenesis conditions with plasmid pPR6821 as a template. Subsequently, papA DNA fragments were digested with BamHI and SalI restriction endonucleases and ligated into plasmid pPR682 to construct malE-papA translational fusion genes. Plasmids containing mutant MBP-pediocin AcH chimeric proteins were identified by colony overlay screening, as explained in the text. Plasmids were named based on the location of the mutation, e.g., plasmid pKN1 contains the K1N mutation (indicated by an asterisk). Abbreviations: ampr, gene encoding β-lactamase; lacIq, gene encoding the lac repressor; lacZ′, gene encoding truncated β-galactosidase; malE, gene encoding MBP; mcs, multiple cloning site; ori, origin of replication; Ptac, tac promoter.