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

he pCAL-n-EK vector, a new addition to the Affinity system, contains the CBP coding sequence followed by the EK cleavage site. This configuration allows efficient cleavage of all sequences at the N terminus of the polypeptide of interest and produces proteins with native amino acid sequence.3 Protein coding sequences are cloned into the pCAL-n-EK vector by LIC, a high-efficiency cloning method that does not require ligation or restriction enzyme digestion. LIC creates a seamless junction4,5,6 between the EK cleavage site and the protein coding sequence. The protein sequence is efficiently cloned into the pCAL-n-EK vector, and protein expression is induced by the addition of isopropyl-thio-D-galactoside (IPTG). The fusion protein is purified in one step using the CBP affinity tag. If desired, the affinity tag can be removed by proteolytic treatment with Stratagene's recombinant Enterokinase, and the recombinant protein of interest can be recovered free of CBP-containing digestion products and EK.

Design of the pCAL-n-EK Vector

figure 1

The pCAL-n-EK vector (figure 1) is the fourth in a series of CBP affinity-tag vectors released by Stratagene.1 It is derived from the pET-11 vector series and contains the lacIq gene for expression of the Lac repressor protein and the hybrid T7 promoter for controlled expression of the inserted protein coding sequence.7,8 The vector is transformed into specialized E. coli strains, such as Epicurian Coli BL21(DE3), which contains an expression cassette for T7 RNA polymerase that is induced in the presence of IPTG, allowing tight control and high-level, induced expression of the inserted protein coding sequence. The promoter is followed by the 26-amino-acid
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