Plasmids with conditional uncontrolled replication behaviour.

摘要

Plasmids are disclosed which carry a regulatable promoter which has been inserted in such a way that transcription from the promoter regulates replication so that increased transcription leads to a substantially increased or uncontrolled plasmid copy number when host microorganisms containing the plasmid are cultivated under conditions securing such increased transcription. The regulatable promoter is preferably lambda PR, the activity of which is controlled by a temperature-sensitive lambda cl repressor. Three types of plasmid are disclosed: one in which lambda PR replaces part of one native replication regulatory gene of the plasmid leading to a plasmid copy number of about 20-40 copies per cell at about 30 DEG C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 36-42 DEG C (a <<type A plasmid>>); another plasmid in which lambda PR has been inserted upstream of the native replication control genes of the plasmid leading to a plasmid copy number of about 3-5 copies per cell at about 30 DEG C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 42 DEG C (a <<type B plasmid>>); and a third plasmid constructed from a type A plasmid in which the deleted native replication gene has been inserted outside the replication region of the plasmid leading to a plasmid copy number of about 0.5-1 copy per cell at about 30 DEG C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 42 DEG C (a <<type C plasmid>>). The plasmids may be used as cloning vectors for the insertion of a fragment of foreign, e.g. eucaryotic, DNA coding for a desired polypeptide. To obtain gene products of the plasmids, host microorganisms to which plasmids of any of the above-mentioned types have been transformed are cultivated at about 30 DEG C to secure a constant, low plasmid copy number during the seeding and multiplication stages of the microorganism, after which the temperature is shifted to about 36-42 DEG C to obtain an uncontrolled plasmid copy number and an amplified amount of gene product. This latter temperature may be maintained until the cells cease to grow, or it may, after a period of time sufficient to obtain a substantial amplification of plasmid DNA, be lowered to the temperature at which the plasmid copy number is constant to secure the continued growth of the host cells while still obtaining amplified amounts of gene product. The gene product is then harvested from the culture.