Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.
The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.
D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase