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

tag for purification of proteins from E. coli extracts1 because of its high affinity for immobilized calmodulin (Kd=10-9). CBP fusion proteins are bound to and eluted from calmodulin affinity matrices at neutral pH using gentle buffer conditions. A relatively low concentration of Ca2+ ions is required for binding (0.2 to 2.0 mM CaCl2), and proteins are efficiently eluted upon removal of the calcium with 2 mM EGTA. The CBP tag is small, (4 kDa) and therefore less likely than some of the larger tags [e.g., the 26-kDa glutathione-S-transferase (GST) and 40-kDa maltose-binding protein (MBP) tags] to affect the biological function of the protein of interest. CBP is efficiently translated in E. coli, and CBP fusion proteins are typically expressed to high levels. In addition, the CBP tag is efficiently phosphorylated by protein kinase A and thus, purified CBP fusion proteins can be readily labeled with isotope and used to probe protein-protein interactions.2

Despite the advantages of using affinity tags for protein purification, there are many proteins for which fusion of nonnatural amino acids at the N- or C-termini adversely affect protein function.3 Most fusion vectors include short recognition sequences for site-specific proteases that allow removal of the fusion tag following purification. Many vectors employ target sequences for proteases that cleave internal to the recognition site and thus, leave at least one extraneous amino acid fused to the protein of interest. The use of the protease enterokinase (EK) has the advantage that it cleaves at the C-terminus of its recognition site, thus when positioned upstream of the protein of interest, N-terminal purification tags can be completely removed without leaving any extraneous amino acid residues on the protein of interest. However, most vectors that employ the use of EK for removal o
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