Nucleic Acids Research, 1992, Vol. 20, No. 7 1785-1791
© 1992
MOLECULAR BIOLOGY |
Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation
Stratagene Cloning Systems 11099 North Torrey Pines Road, La Jolla, CA 92037, USA
* To whom correspondence should be addressed
Received August 27, 1991. Revised February 10, 1992. Accepted February 10, 1992.
Eukaryotic expression vectors designed to produce E. coll Lac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacl) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lad gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified Lacl protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while Introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the Lacl protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacl behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-termlnus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modified lacl gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.
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