RUNAWAY-REPLICATION PLASMIDS AS TOOLS TO PRODUCE LARGE QUANTITIES OF PROTEINS FROM CLONED GENES IN BACTERIA

Here we review the properties and uses of runaway-replication vectors, a class of versatile plasmids discovered and developed in Escherichia coli. They are based on the IncFII plasmid, R1, in which an antisense RNA (CopA RNA) negatively controls the formation of a protein that is rate-limiting for replication. The copy number of the plasmid is determined by the balance between the rates of formation of CopA RNA and RepA mRNA. A small increase in the rate of formation of the latter drastically reduces the rate of formation of CopA RNA due to convergent transcription, which may lead to a total loss of copy number control (runaway replication), resulting in massive DNA amplification, and plasmid copy numbers up to 1000 per genome. Since this amplification occurs in the presence of protein synthesis, the protein that is encoded by a cloned gene can also be amplified, and may constitute 10-50% of the total protein.