( the pESP-2 vector results in a seamles...)New Yeast Cloning System for Producing Proteins with Native Amino Acid,,,Sequences

the pESP-2 vector results in a seamless junction of the enterokinase site and the initiation codon of the gene to be expressed. The pESP-2 vector is supplied in a form ready for LIC cloning, with 12- and 13-nucleotide, single-stranded overhangs. The gene of interest is prepared for PCR amplification and LIC cloning by using primers that include 12- and 13-nucleotide sequences that are complementary to the 5 single-stranded overhangs of the pESP-2 vector. Following PCR amplification, the PCR product is treated with Pfu DNA polymerase in the presence of dATP. In the absence of dTTP, dGTP and dCTP, the 3- to 5-exonuclease activity of Pfu DNA polymerase removes 12 to 13 nucleotides at the respective ends of the PCR product. This activity continues until the first adenine is encountered, when the polymerase activity of the enzyme becomes dominant, producing a DNA fragment with 5-extended single-stranded tails that are complementary to the single-stranded tails of the pESP-2 vector. The insert anneals to the vector without further enzymatic treatment and is ready for transformation into XL10-Gold ultracompetent cells.

Protein Expression and Purification

Figure 3A & 3B

We have expressed and purified a number of recombinant proteins in S. pombe, using the pESP-2 vector and the LIC cloning strategy to create the constructs. The recombinant proteins include human Elk1, Krs2, MEK1^WT and the chicken calmodulin gene product. The yields for GST-Elk1 and GST-Krs2 fusion proteins were relatively low, approximately 1 mg or less per liter, while the GST-MEK1^WT
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