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- Title
Extracellular expression of human epidermal growth factor encoded by an Escherichia coli K-12 plasmid stabilized by the ytl2-incR system of Salmonella typhimurium.
- Authors
Wong, D K-H; Lam, K H E; Chan, C K P; Wong, Y C V; Wong, W K R; Hackett, J
- Abstract
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 >10[SUP4]-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 10[SUP8] cells at harvest, compared to a figure of ca 3 μg per 10[SUP8] 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.
- Subjects
PLASMIDS; SALMONELLA typhimurium
- Publication
Journal of Industrial Microbiology & Biotechnology, 1998, Vol 21, Issue 1/2, p31
- ISSN
1367-5435
- Publication type
Article
- DOI
10.1038/sj.jim.2900557