To the top

Page Manager: Webmaster
Last update: 9/11/2012 3:13 PM

Tell a friend about this page
Print version

A Novel Nonantibiotic, lg… - University of Gothenburg, Sweden Till startsida
To content Read more about how we use cookies on

A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae

Journal article
Authors Manuela Terrinoni
Stefan Nordqvist
Susanne Källgård
Jan Holmgren
Michael Lebens
Published in Applied and Environmental Microbiology
Volume 84
Issue 4
ISSN 0099-2240
Publication year 2018
Published at Institute of Biomedicine, Department of Microbiology and Immunology
Language en
Keywords plasmid maintenance, Gram-negative bacteria, essential genes, complementation, toxin-b-subunit, antibiotic-resistance, vaccine development, protein, gene, construction, atherosclerosis, auxotrophy, bacteria, increase, Biotechnology & Applied Microbiology, Microbiology
Subject categories Microbiology, Medical Biotechnology


Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro) lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30 degrees C but not at 37 degrees C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39 degrees C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli. The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation. IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

Page Manager: Webmaster|Last update: 9/11/2012

The University of Gothenburg uses cookies to provide you with the best possible user experience. By continuing on this website, you approve of our use of cookies.  What are cookies?