A novel non-antibiotic, lgt-based selection system for stable maintenance of expression vectors in Escherichia coli and Vibrio cholerae.

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 biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in E. coli and other gram negative organisms. The lgt gene was deleted from E. coli and complemented by the V. cholerae derived gene provided in trans on a temperature sensitive plasmid allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. A reciprocal construction was done 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 new-generation oral cholera vaccines. Overall, the lgt system described confers extreme stability on expression plasmids and the 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 encoded on autonomously replicating DNA elements called plasmids. These are usually inherently unstable and rapidly lost. This c...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research