( xonuclease activity of Pfu DNA polymera...)Expression and Purification of Recombinant Proteins That Have Native Amino,,,Acid Sequence

xonuclease activity of Pfu DNA polymerase removes at least 12 and 13 nucleotides at the respective 3' ends of the PCR product. This activity continues until the first adenine is encountered, producing a DNA fragment with 5'-extended single-stranded tails that are complementary to the single-stranded tails of the pCAL-n-EK vector. The vector and insert DNA are combined, allowed to anneal at room temperature and transformed into highly competent bacterial host cells. The resultant colonies can then be screened for the desired insert by PCR amplification.

Efficiency of Cloning into the pCAL-n-EK Vector

To test the efficiency of cloning into the LIC-prepared pCAL-n-EK vector, the sequence encoding c-Jun N-terminal kinase (JNK)¹⁰ was PCR amplified using gene specific primers containing the 12 and 13-nucleotide sequences complementary to those in the pCALnEK vector. The 1280-bp JNK sequence that resulted from PCR amplification was gel purified, treated with Pfu DNA polymerase at 72C in the presence of dATP and annealed to the pCALnEK vector containing complementary single-stranded tails. The annealing reaction was transformed into Epicurian Coli XL1-Blue supercompetent E. coli cells and spread onto LB plates containing ampicillin. Of the thousands of colonies that resulted from several transformations, 42 were screened by PCR, and 41 (98%) were found to contain the JNK insert. The accuracy of the LIC cloning method was confirmed by sequence analysis of the regions flanking the N terminus and C terminus of the inserted gene.

LIC cloning was also performed with 50 ng of prepared DNA coding for the kanamycin gene annealed to 30 ng of prepared pCAL-n-EK vector DNA. Onetenth of the reaction was transformed into XL1Blue supercompetent cells, resulting in approximately 800 colony forming units (cfu); 100% were ampicillin and kanamycin
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