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

f N-terminal purification tags require the use of cloning schemes (e.g., restriction sites) that constrain the codon choice immediately downstream of the EK cleavage site, and thus predetermine the N-terminal amino acid sequence of the protein of interest.

We recently described the CBP fusion vector pCAL-n-EK4 in which the ligation-independent cloning (LIC) method5 is employed such that any amino acid coding sequence can be seamlessly fused immediately downstream of the EK cleavage site, thereby allowing the recovery of proteins from EK cleavage reactions that have native N-terminal amino acid sequence. In addition to the ability to create seamless fusions between the CBP purification tag and the protein coding sequence of interest, cloning by LIC is highly efficient. Cloning efficiencies of 2 x 105 cfu/g vector are routinely achieved using as little as 25 ng PCR-generated insert; thus, enough insert for several cloning reactions may be generated with as little as 10 cycles of PCR amplification.4

There is an inherent drawback to the use of CBP fusions for certain downstream applications for which purified proteins are employed to probe protein-protein interactions. The ubiquitous presence of calmodulin in eukaryotic cells, in addition to the presence of endogenous calmodulin-binding proteins can, under some conditions, contribute to background in such applications. Specifically, immunoaffinity-type pull-down experiments in which CBP-fusions are recovered from crude eukaryotic extracts by immunoprecipitation with calmodulin affinity resin often result in the recovery of calmodulin-binding proteins.

Features of the pCAL-n-FLAG Vector

Figure 1

The pCAL-n-FLAG vector (Figure 1) is the fifth in a series of CBP affinity-tag vecto
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