EXTRACELLULAR EXPRESSION OF HUMAN EPIDERMAL GROWTH-FACTOR ENCODED BY AN ESCHERICHIA-COLI K-12 PLASMID STABILIZED BY THE YTL2-INCR SYSTEM OFSALMONELLA-TYPHIMURIUM

Citation
Dkh. Wong et al., EXTRACELLULAR EXPRESSION OF HUMAN EPIDERMAL GROWTH-FACTOR ENCODED BY AN ESCHERICHIA-COLI K-12 PLASMID STABILIZED BY THE YTL2-INCR SYSTEM OFSALMONELLA-TYPHIMURIUM, Journal of industrial microbiology & biotechnology, 21(1-2), 1998, pp. 31-36
Citations number
17
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biothechnology & Applied Migrobiology
Volume
21
Issue
1-2
Year of publication
1998
Pages
31 - 36
Database
ISI
SICI code
Abstract
A plasmid stabilization system, active in high copy-number plasmids, w as cloned from the large resident plasmid, pSLT, of Salmonella typhimu rium, The ytl2 gene, together with a 249-bp region (termed incR) downs tream of the gene, imparted > 10(4)-fold stability to a pBR322-based p lasmid, The ytl2-incR region was then used to stabilize a recombinant plasmid carrying the human epidermal growth factor gene (with the Esch erichia coli K-12 ompA signal sequence), behind the lacUV5 promoter, I n shake flask tests to optimize expression of human epidermal growth f actor, loss of recombinant plasmid was <1% when growth (both before an d after induction with isopropyl-beta-D-galactopyranoside) took place even in the absence of antibiotic selection, and the specific activity of secreted human epidermal growth factor was ca 20 mu g per 10(8) ce lls at harvest, compared to a figure of ca 3 mu g per 10(8) cells when a comparable plasmid, but devoid of the ytl2-incR region, was employe d, as outgrowth of plasmid-free cells after induction severely comprom ised the specific activity of the secreted product.