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High-Level Protein Expression, One-Column Purification, and FLAG,,,Epitope Tagging in E. coli


Seamless insertion and one-step purification from a more versatile vector

High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli

Katherine Felts Denise Wyborski John Bauer Peter Vaillancourt
Stratagene

Stratagene has improved the Affinity LIC cloning kit with the new pCAL-n-FLAG vector. This new E. coli cloning and expression vector is designed for consistent, high-level production and one-step purification of expressed proteins. Similar to its predecessor pCAL-n-EK, pCAL-n-FLAG has the same features with the addition of the FLAG epitope ll ll as an N-terminal fusion with the protein of interest. The FLAG epitope is useful to those scientists desiring a small antibody recognition site on their protein of interest. This vector will be of particular interest to scientists who wish to use calmodulin affinity-purified proteins to study protein-protein interactions by pull-down immunoprecipitation experiments using eukaryotic cell extracts, or who wish to probe lambda cDNA expression libraries for interacting proteins. In both cases, the presence of endogenous calmodulin-binding proteins, which are ubiquitous in all eukaryotic cell types, may contribute to background. The calmodulin-binding peptide (CBP) purification tag can be removed from the recombinant protein by thrombin digestion. If desired, the FLAG tag can be removed by digestion with enterokinase, leaving protein of native amino acid sequence.

Purification of recombinant proteins has been greatly simplified in recent years due to the availability of expression vectors that allow fusion of the protein coding sequence of interest to short peptide sequences, or larger proteins, enabling the affinity purification of the fusion protein from crude preparations. The 26-amino acid CBP is an attractive fusion
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