An Escherichia coli expression vector was constructed for the production-scale fermentation of recombinant bovine somatotropin (rBST). Gene expression is regulated by a spontaneous increase in copy number at a constant low temperature without the need for an external inducer. This vector, designated pURA-4, contains the ampicillin resistance gene, the replication origin from pBR322, the R1 temperature-inducible runaway replicon, and a gene encoding rBST. Optimized rBST expression levels of >35% total cell protein were achieved at a constant 28degreesC. Shake-flask analysis of pURA-4 shows that the copy number spontaneously increases approximately 6-fold during rBST production. Investigation into the mechanism of pURA-4 spontaneous runaway shows that the increase in copy number is directed by the pBR322 ori and not by the R1 replicon. Although the R1 temperature-inducible replicon does not mediate spontaneous runaway, it does have a positive effect on rBST expression. Copy number analysis also confirmed the stability of pURA-4 spontaneous runaway from the shake-flask scale through the production scale.