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

rs released by Stratagene. It is derived from the pET-11 vector series and contains the lacI q gene for expression of the Lac repressor protein and the hybrid T7 promoter for controlled expression of the inserted protein coding sequence.8 The vector is transformed into specialized E. coli strains, such as Epicurian Coli BL21(DE3), which contain 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 CBP affinity tag followed by a thrombin cleavage site, the FLAG epitope, an enterokinase cleavage site, the multiple cloning site (MCS), and the T7 transcriptional terminator.

LIC Cloning of JNK

Figure 2

Highly efficient, directional cloning of the desired insert into the pCAL-n-FLAG vector is by ligation-independent cloning (LIC), a method that does not require ligation or restriction enzyme digestion (Figure 2, Panel A). The pCAL-n-FLAG vector is pretreated to create noncomplementary, 12- and 13-nucleotide single-stranded tails at the two 5 ends of the vector. These noncomplementary tails prevent the vector from recircularizing. The insert DNA is prepared by PCR amplification with gene-specific primers that include 12- and 13-nucleotide sequences at the 5 ends that are complementary to the pCAL-n-FLAG vector single-stranded tails, thus allowing for directional cloning. Following PCR amplification , the PCR product is purified, then treated with Pfu DNA polymeras
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