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New Yeast Cloning System for Producing Proteins with Native Amino Acid,,,Sequences

es of PCR) and Stratagenes high-fidelity Pfu DNA polymerase. After purification of the PCR product, a simple, quick Pfu DNA polymerase treatment of the PCR product replaces restriction enzyme digestion; annealing of insert and vector fragments with specific ends ensures high cloning efficiency. Cloning of the PCR product is directional, and more than 80% of the transformants contain an insert in the correct orientation.

The PCR products are cloned into the pESP-2 vector immediately downstream of the GST affinity tag and the enterokinase (EK) recognition site, resulting in a seamless junction between the EK cleavage site and the initiation codon (ATG) of the gene to be expressed. After expression in S. pombe, the GST fusion protein can be purified from crude cell extracts using a single-step purification procedure based on the selective affinity of the GST peptide for glutathione immobilized on resin. Following the single-step purification procedure, the GST affinity tag may be removed by proteolytic cleavage with EK to yield polypeptides with native amino acid sequence. Cleavage with thrombin yields the recombinant protein fused with the FLAG epitope for future analysis of the protein.

The ESP LIC cloning kit contains pretreated, ready-to-use pESP-2 vector, Pfu DNA polymerase and 10X reaction buffer, Epicurian Coli XL10-Gold ultracompetent cells, forward and reverse primers for sequencing and PCR amplification and other LIC cloning reagents and control plasmids. The ESP LIC cloning and protein purification kit includes all of these components plus S. pombe competent cells (strain SP-Q01), YES media for vegetative growth, Edinburgh minimal media (EMM) for induction, acid-washed glass beads and GST affinity resin.

Ligation-Independent Cloning

figure 2

The LIC strategy (figure 2) using
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