A self-inducing runaway-replication plasmid expression system utilizing the Rop protein.
A highly efficient prokaryotic expression system has been developed that produces proteins at levels exceeding 150 micrograms/ml of culture medium. The system consists of a temperature-sensitive-copy-number plasmid that carries the rop gene and promoter downstream from the trp promoter. Any sequence cloned into the PvuII site of the rop gene alters Rop protein activity and causes lethal runaway plasmid DNA replication. This plasmid replication can be suppressed in trans by complementation with a similar wild-type plasmid. Cells harboring both plasmids are quite stable, and induction of plasmid DNA synthesis occurs only after cells are grown for several generations under conditions that lead to the loss of the trans-acting repressor. Large amounts of Rop fusion proteins accumulate in the cell as the trp operon is gradually induced via repressor titration. All chimeric proteins accumulate as insoluble aggregates, and are therefore easily purified. They can be solubilized using relatively mild conditions, and the partially purified proteins are highly amenable to cleavage by chemical methods. Using this system we have made Rop fusions with the HIV Tat protein, the herpes simplex virus type-2 38K protein, and Chinese hamster metallothionin.
Giza
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Department of Biology, Johns Hopkins University, Baltimore, MD 21218.
Huang
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003087:Colicins / Q000235:genetics; D004247:DNA; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*
D001426:Bacterial Proteins; D003087:Colicins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C054243:Rop protein, ColE1 plasmid; D004247:DNA