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Expression and Purification of Recombinant Proteins That Have Native Amino,,,Acid Sequence

One column purification of protein with native amino acid sequence

Denise L. Wyborski * John C. Bauer * Barbara McGowan * Joseph A. Sorge * Peter Vaillancourt
Stratagene Cloning Systems, Inc.

Stratagene has improved the Affinity protein expression and purification system by the addition of the pCAL-n-EK vector. This E. coli cloning and expression vector is designed for consistent, high-level production and one-step purification of expressed proteins. A ligation-independent cloning (LIC) strategy is used to obtain high-efficiency cloning of the desired protein sequence into the pCAL-n-EK vector. LIC creates seamless cloning junctions between the protein coding sequence of interest and the recognition target for the site-specific protease, enterokinase (EK). The EK target sequence is located between the calmodulin-binding peptide (CBP) purification tag and the N terminus of the expressed protein. Because EK cleaves at the C terminus of its recognition target, which is also the N terminus of the inserted polypeptide sequence, cleavage of fusion proteins produced in the pCAL-n-EK vector yields the desired fusion partner free of any extraneous amino acids derived from the fusion tag. Therefore, cleavage results in the production of native protein.

Stratagene's Affinity protein expression and purification system uses the 26-amino-acid CBP sequence as an affinity tag for purifying recombinant proteins from crude cell lysates with a single pass through calmodulin affinity resin.1,2 The CBP purification system is an excellent alternative to other affinity-tag systems because of its gentle binding and elution characteristics. Another advantage of this system is the small size of the CBP affinity tag (4 kDa). In comparison to other larger affinity tags, the CBP tag is less likely to affect the physical characteristics of the protein of interest.

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