Purification of Rts1 RepA protein and its characterization

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1. Purification of RepA protein. The repA gene, encoding RepA protein that is essential for the replication of Rts1, was inserted into a vector plasmid pKK223-3, thereby expressing repA under the control of tac promoter in E. coli JM103. RepA in a crude lysate obtained from 10 liters culture of the cells induced by IPTC was purified through chromatographies using CM-sephadex, phosphocellulose and Affigel blue columns. The amino acid composition of the purified RepA was in agree with the composiion deduced from the repA sequence.2. Binding of RepA to DNA (examined by DNase I protection assay).The purified RepA was confirmed to bind strongly to the immediately upstream region of promoter of repA(which would be an operator) and a 10-bp portion between incII and GATC box besides incI and incII repeated sequences. The 10-bp region would be a possible site of origin of Rts1 replication.3. Isolation of ori(Rts1). A mini-Rts1 subregion that spans the coordinates 1441 to 1194 was capable of initiating relpication when RepA was supplied in trans from mini-Rts1 plasmid in vivo study. In the ori(Rts1), incII sequences along with GATC and DnaA boxes are contained.4. Deletion of C terminus of RepA. A series of repA mutants that encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl terminal six amino acids were constructed. These modified RepA could not activate ori(Rts1) at all. One of the RepA derivatives, however, maintained a weak but evident incompatibility toward mini-Rts1 plasmid, which may be caused by binding of the RepA to operator of repA.