Overproduction and Operator DNA-Protein Blotting of R100 Mutant MerR from Shigella flexneri

Wild-type and four mutant R100 merR genes were cloned and the proteins overproduced under tac promoter control of pKK223-3. His118Ala, Cys117Ser, Cys126Ser, and wild-type MerR were successfully overproduced although amino-terminal 14 amino acids deletion mutant MerR was not successful. The amount of overproduced wild-type MerR protein as well as other mutant MerR was between 15%-20% of the total protein. The protein was able to be purified up to 95% homogeneity. Specific DNA-protein blotting experiments showed that the 95 bp operator containing DNA fragment could bind to Cys126Ser, His118Ala, and wild- type MerR, but not to Cys117Ser. These results were consistent with the previously reported complementation experiment results that His118Ala, Cys126Ser, and wild-type MerR could repress the mer operon but Cys117Ser could not.