Keywords: hEGF; plasmid pSLT; ytl2-incR stabilization
Springer Online Journal Archives 1860-2000
Process Engineering, Biotechnology, Nutrition Technology
A plasmid stabilization system, active in high copy-number plasmids, was cloned from the large resident plasmid, pSLT, of Salmonella typhimurium. The ytl2 gene, together with a 249-bp region (termed incR) downstream of the gene, imparted 〉104-fold stability to a pBR322-based plasmid. The ytl2-incR region was then used to stabilize a recombinant plasmid carrying the human epidermal growth factor gene (with the Escherichia coli K-12 ompA signal sequence), behind the lacUV5 promoter. In shake flask tests to optimize expression of human epidermal growth factor, loss of recombinant plasmid was 〈1% when growth (both before and after induction with isopropyl-β-d-galactopyranoside) took place even in the absence of antibiotic selection, and the specific activity of secreted human epidermal growth factor was ca 20 μg per 108 cells at harvest, compared to a figure of ca 3 μg per 108 cells when a comparable plasmid, but devoid of the ytl2-incR region, was employed, as outgrowth of plasmid-free cells after induction severely compromised the specific activity of the secreted product.
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