Use of gene fusions and protein - protein interaction in the isolation of a biologically active regulatory protein : The replication initiator protein of plasmid R 6 K ( protein tagging / subunit interaction / protein sequence

The initiation of DNA replication of plasmid R6K is triggered by a 35-kldodalton 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 f8-galactoside. Using an affinity column that is specific for P-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 P-galactosidasefused 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. Initiation of replication of chromosomes that replicate in the Cairns-type mode is triggered by the interaction of replication initiator proteins with specific sequences on the DNA (1). The biochemical elucidation of the initiation process still remains a major goal in molecular biology. Purification of replication initiator proteins, obviously, is a key step in the biochemical dissection of the initiation process. We have used the drug-resistance plasmid R6K (2), which replicates in the Cairns-type configuration from multiple origins of replication (3-5), as a model system to study replication initiation. The replication of R6K is triggered by a 35-kilodalton (kDa) plasmid-encoded initiator protein (6-8). The apparent lability and lack of an adequate assay procedure posed serious problems during previous attempts to purify this protein. We have previously reported that the initiator can be tagged by fusion with the 89-amino-acid-long a-donor peptide of f3-galactosidase to yield a fused protein that retains its ability to initiate DNA replication in vivo (9). However, this approach failed to yield a homogeneous preparation of stable hybrid-initiator protein. In this communication we describe the fusion of the replication initiator gene of R6K with a larger B-galactosidase-coding sequence in such a way that the 3' end of the initiator gene is covalently attached to the ninth amino acid codon from the NH2 terminus of the lacZ cistron, in the correct translational frame. We further show that the fusion apparently stabilizes the initiator protein without destroying its biological activity, as measured by in vivo and in vitro DNA replication initiated from the region of replication origin y. Using an affinity matrix that specifically retains B-galactosidase, we have demonstrated the rapid purification of the hybrid initiator to near homogeneity. Exploiting the multimeric structure of the initiator, we have also shown that the nonfused initiator protomer can be rapidly isolated by affinity column chromatography because of subunit interaction between the fused and the nonfused initiator proteins produced from plasmids carrying a second nonfused copy of the initiator gene. MATERIAL AND METHODS Bacterial and Plasmid Strains. The Escherichia coli strain MC1000 (ara D139, A (ara, leu), 7697 A lac X 74, galU galK, StrA) was obtained from Malcom Casadaban (University of Chicago). The lacZ fusion vectors pORF5 and pMLB1031 were obtained from M. L. Berman through C. Turnbough (University of Alabama at Birmingham). The plasmid pJG100, that contains the initiator protein cistron of R6K in a BamHI fragment, has been described (9). Construction of Recombinant DNA Clones. These were carried out by using standard procedures as described (9). Preparation of the Affinity Matrix. The diaminohexane linker was attached to cyanogen bromide-activated Sepharose 4B and then succinylated with succinic anhydride. The substrate paminophenyl-f3-D-thio-galactoside was then attached to the succinyl diaminohexane arm with water-soluble carbodiimide. The entire procedure has been described (10). The chemicals used and ready-made affinity matrix are available from Sigma. Purification of ,-Galactosidase-Tagged Protein. Bacterial cells (strain MC1000) containing the plasmid pJG10 or pJGll (see Results) were grown in standard Luria broth to a cell density of 2-5 x 108 cells per ml and then were harvested. All subsequent operations were carried out at 4°C. The cell pellet was then suspended in a suitable volume of buffer B (0.2 M Tris-HCl, pH 7.6/0.25 M NaCl/0.01 M magnesium acetate/10 mM 2-mercaptoethanol/5% glycerol; 10-ml volume per liter of culture). Lysozyme was added to 0.2 mg/ml and the suspension was kept on ice for 30 min. The cell suspension was frozen at -70°C and thawed quickly in a 30°C water bath, taking care not to allow the temperature of the thawing suspension to rise above 4°C. After brief sonication to reduce the viscosity, phenylmethylsulfonyl fluoride was added to 1 mM. The lysate was Abbreviations: NH2PhSGal-Sepharose, p-aminophenyl-f3-D-thio-galactosidyl succinyldiaminohexyl-Sepharose; kDa, kilodalton(s). * To whom reprint requests should be addressed. 6848 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 6849 clarified by centrifugation at 35,000 rpm in a type 60 Ti rotor (Beckman) for 30 min at 40C. The f-galactosidase activity in the cleared lysate was determined as described (11). Nucleic acids were precipitated by adding slowly 0.1 vol of 30% (wt/vol) streptomycin sulfate. Precipitated material was removed by centrifugation. An equal volume of 80% (of saturation) ammonium sulfate solution was added slowly with stirring. The precipitate was collected by centrifugation and the supernatant was checked for (3-galactosidase activity to make sure that it had <10% of the original activity. The supernatant was then discarded. The pellet was then suspended in buffer D (0.25 M NaCl/10 mM Tris HCl, pH 7.6/10 mM MgCl2/1 mM EDTA/ 10 mM 2-mercaptoethanol/0. 1% Triton X-100) and dialyzed against 100 vol of the same buffer for 3 hr. The dialysate containing 90% of the original /-galactosidase activity was passed through a 10-ml bed volume of p-aminophenyl-f-D-thio-galactosidyl succinyldiaminohexyl-Sepharose (NH2PhSGal-Sepharose) in a column at 40C. Although -90% of the applied protein appeared in the flow-through, <10% of the input (-galactosidase activity was detectable in that fraction. The column was washed with 10 bed vol of buffer D, followed by 5 bed vol of buffer D without Triton X-100. The protein was eluted from the column with 0.1 M sodium borate (pH 10) and promptly precipitated with ammonium sulfate. NH2-Terminal Amino Acid Sequence. The NH2-terminal sequence was analyzed in a Beckman sequencer by using a 0.55 M Quadrol program and the resulting phenylthiohydantoin amino acids were analyzed by high-pressure liquid chromatography as described by Vanaman and co-workers (12). Two-Dimensional Gel Electrophoresis. This was carried out as described (13). In Vitro DNA Replication. Cell extracts of E. coli K12 (strain W3110) were prepared as described (14). The cell extract was supplemented with various amounts of purified ,B-galactosidase-tagged initiator protein, purified supercoiled DNA, and other components as described in the legend to Fig. 3.