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Applied and Environmental Microbiology, October 2002, p. 5051-5056, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5051-5056.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

Jacob Glenting,1* Søren M. Madsen,1 Astrid Vrang,1 Anders Fomsgaard,2 and Hans Israelsen1

Department of Lactic Acid Bacteria, Biotechnological Institute, DK-2970 Hørsholm,1 Department of Virology, Statens Serum Institut, DK-2300 Copenhagen, Denmark2

Received 17 April 2002/ Accepted 16 July 2002

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363{Delta}thr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363{Delta}thr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.

* Corresponding author. Mailing adress: Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. Phone: (45) 45 16 04 44. Fax: (45) 45 16 04 55. E-mail: jag{at}bioteknologisk.dk.

Applied and Environmental Microbiology, October 2002, p. 5051-5056, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5051-5056.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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