PURIFICATION OF pKYM-ENCODED REPK, A PROTEIN REQUIRED FOR THE INITIATION OF PLASMID REPLICATION

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Replication of pKYM requires the plasmid-encoded RepK protein, which specifically binds to the replication origin of the plasmid. This binding was easily detected in a gel-shift assay. We have used this characteristic to purify RepK protein, to near homogeneity. The repK gene was placed under the control of the phage T7 RNA polymerase promoter on a plasmid. Upon induction of T7 RNA polymerase, Escherichia coli clones containing the recombinant plasmid overproduced the RepK protein, which was then purified in significant quantities. The molecular weight of the RepK protein under denaturing conditions is 36, 000, which is consistent with the size predicted from the DNA sequence data. The N-terminal amino acid sequence determined for the purified RepK matches that deduced from the DNA sequence. RepK protein appears to bind to the replication origin as a monomer with a dissociation constant (K_d) of 2.7×10-9M.