High-Level Protein Expression, One-Column Purification, and FLAG,,,Epitope Tagging in E. coli
... Seamless insertion and one-step purification from a more versatile ...Stratagene has improved the Affinity LIC cloning kitwit...Purification of recombinant proteins has been greatly simplified in re...
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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