spontaneous runaway vector for production-scale expression of bovine somatotropin from Escherichia coli

• Published in: Applied microbiology and biotechnology Vol. 58; no. 1; pp. 84 - 88
• Main Authors: Trepod, C.M, Mott, J.E
• Format: Journal Article
• Language: English
• Published: Berlin Springer 2002; Springer Nature B.V
• Subjects: ampicillin; Biological and medical sciences; Biotechnology; bovine somatotropin; E coli; Escherichia coli; Fermentation; Fundamental and applied biological sciences. Psychology; Gene expression; genes; Genetic engineering; Genetic technics; growth hormone; Low temperature; Methods. Procedures. Technologies; Modification of gene expression level; replication origin; somatotropin; temperature; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Bovine; Copy number; Escherichia coli; Somatotropin hormone; Gene expression; Plasmid; Vertebrata; Mammalia; Production; Bacteria; Artiodactyla; Recombinant protein; Ungulata; Enterobacteriaceae
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-001-0859-6

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 28 degrees C. 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.