Construction of a runaway vector and its use for a high-level expression of a cloned human superoxide dismutase gene

Summary

A runaway cloning vector pR3 from plasmid pBEU17 has been constructed by introducing three deletions (ΔI, ΔH and ΔEHi), multiple cloning sites and transcription terminator sequences. Introduction of the deletion ΔH restored a drastic plasmid amplification at high temperature (37° C). This suggested the presence of unknown sequences around the ΔH region which repressed runaway replication. To examine the usefulness of a pR3 runaway vector on the high-level expression of a cloned gene, a pRT8 plasmid containing a cloned human superoxide dismutase (SOD) gene in the pR3 was constructed. A host with tolerance to high copy numbers of pRT8 was selected, and the cultural conditions required for high-level expression were investigated. It was shown that the copy number of pRT8 increased in the stationary phase even at 30° C, and that cells carrying an excess number of plasmid did not support the runaway replication, yielding less SOD protein. SOD synthesis in the production culture lasted for about 24 h after the thermal shift, though viability was soon lost. The maximum yield was determined as approximately 20% of total cellular protein. This was 12-fold higher than that of a vector having the ColE1 plasmid replicon.