title: Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.
abstract: High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.
authors.affiliation: Laboratory of Molecular Ecology and Biotechnology, National Research Council, Institute for Marine and Coastal Environment (IAMC-CNR), Detached Unit of Capo Granitola, Torretta Granitola (TP), Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Tagliavia
authors.affiliation: Laboratory of Molecular Ecology and Biotechnology, National Research Council, Institute for Marine and Coastal Environment (IAMC-CNR), Detached Unit of Capo Granitola, Torretta Granitola (TP), Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Cuttitta
mesh_terms: D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D011994:Recombinant Proteins
keywords: expression vectors; recombinant proteins; selection of expressing cells; translational coupling