Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins.
We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production.
Bouquin
Thomas
T
Maxygen ApS, Agern Allé 1, DK-2970 Hoersholm, Denmark. tbo@maxygen.dk
Rasmussen
Poul Baad
PB
Bertilsson
Per-Anders
PA
Okkels
Jens Sigurd
JS