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

CBP affinity tag1,2 and the 5-amino-acid EK cleavage target.3 To provide maximal cloning and expression flexibility, Stratagene has refined the LIC method for cloning inserts into the pCAL-n-EK vector such that there are no constraints on the N-terminal amino acid of the protein coding sequence. The majority of proteins that are expressed in yeast and higher eukaryotes have their N-terminal methionines removed during a posttranslational processing event, an event that is often required to obtain a functional protein.9 This cloning flexibility will be an important feature for researchers who are interested in cloning protein domains, where an N-terminal methionine would be unwanted.

LIC of the Protein Coding Sequence

figure 2

Highly efficient, directional cloning of the desired insert into the pCAL-n-EK vector is by LIC, a method that does not require ligation or restriction enzyme digestion (figure 2).4,5,6 The pCAL-n-EK vector is pretreated to create non-complementary, 12 and 13-nucleotide single-stranded tails at the two 5 ends of the vector. These non-complementary tails prevent the vector from recircularizing, virtually eliminating nonrecombinant plasmids. 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-EK vector single-stranded tails, thus allowing for directional cloning. Following PCR amplification, the PCR product is purified and treated with Pfu DNA polymerase in the presence of dATP. In the absence of dTTP, dGTP and dCTP, the 3 to 5-e
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