Report on a Workshop: Plasmid Cloning Vehicles

The first three contributions addressed the problem of designing plasmid vectors for the expression of foreign proteins in E. coli. All three used the principle that, to reduce the genetic and metabolic load to the cell, low or moderate copy-number plasmids should be used, and in two cases higher copy numbers were induced late in the growth phase. Yarraiton (Celltech) reported on experiments to express calf met-prochymosin. Initial experiments with the high copy ampicillin-resistance plasmid (based on pAT153) using a trp promoter and T7 terminator gave 5% loss/generation which could not be stabilized by ampicillin selection during scale-up. Repressed levels of production even in the presence of the pSClOl par locus caused instability because the trp promoter was not fully repressed when it was on the high copy plasmid. Vectors were therefore constructed in which the primer RNAII transcription could be brought under control of various inducible promoters, in particular, bacteriophage P under the control of the Acl857 gene product. In the presence or the pSClOl par region these plasmids carrying the trp-controlled prochymosin gene were stable at 30°C in the absence of antibiotic selection, having a copy number of 5/genome.