( d onto a nitrocellulose filter and subj...)High-Level Protein Expression, One-Column Purification, and FLAG,,,Epitope Tagging in E. coli

d onto a nitrocellulose filter and subjected to western blot analysis with an anti-c-Jun antibody (Transduction Laboratories, Lexington, KY) by standard procedures. As seen in Figure 5, Panel A, Lanes 3 and 4, roughly equivalent amounts of FLAG-JNK are observed on the Coomassie Blue stained gel. In Figure 5, Panel B, an anti-c-Jun-specific band with a gel mobility corresponding to the expected molecular weight is specifically immunoprecipitated from HeLa nuclear extract with the M2-FLAG-JNK gel (Lane 4).

Figure 5

Conclusion

Stratagene has improved the Affinity LIC cloning kit by the addition of a more versatile vector, pCAL-n-FLAG. This vector has the same features as its predecessor pCAL-n-EK, such as seamless insertion by LIC, and one-step purification using the CBP purification tag and calmodulin resin. The vector has been improved by the addition of the FLAG epitope between the CBP purification tag and the protein of interest. This enables the researcher to remove the CBP from the purified protein while retaining an antibody recognition site. This feature facilitates the use of the recombinant protein of interest in protein-protein interaction experiments using eukaryotic cell extracts. Both the CBP and FLAG fusions can be removed from the purified protein by digestion with enterokinase, liberating protein of native amino acid sequence.

REFERENCES

1. Stofko-Hahn, R.E., Carr, D.W., and Scott, J.D. (1992) FEBS Lett. 302: 274-278.

2. Simcox, T.G., et al. (1995) Strategies 8: 40-43.

3. LaVallie, E.R., et al. (1993) J. Biol. Chem. 268: 23311-23317.

4. Wyborski, D.L., et al. Strategies 10: 15-18.

5. Aslanidis,
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