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Mammalian and Bacterial Expression in One Vector

(lane 4), approximately 50% of the CBP fusion tag was cleaved after a 6-hour incubation at room temperature (lane 5) and approximately 95% of the fusion tag was cleaved after 24 hours (lane 6).

Conclusions

The pDual expression vector was developed to allow simultaneous expression of proteins in prokaryotic and mammalian systems. The vector contains a hybrid bacterial promoter for inducible bacterial expression, the CMV promoter and enhancer region for constitutive mammalian expression and tandem consensus sequences for optimal translation initiation in both systems. The cloning site is compatible with the novel Seamless cloning technique.3,4 The CBP-affinity tag is located downstream of the cloning site, allowing simple detection and purification of a protein of interest. The CBP-affinity tag can be removed from the fusion protein via the thrombin cleavage site that immediately precedes the tag.

REFERENCES

  1. Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. USA 71: 1342-1346.

  2. Kozak, M. (1986) Cell 44: 283-292.

  3. Padgett, K.A., and Sorge, J.A. (1996) Gene 168: 31-35.

  4. Padgett, K.A., and Sorge, J.A. (1996) Strategies 9: 14-16.

  5. Chalfie, M., et al. (1994) Science 263: 802-805.

  6. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

* Patent pending


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