( One-column protein purification and n...)New Yeast Cloning System for Producing Proteins with Native Amino Acid,,,Sequences

One-column protein purification and native eukaryotic amino acid sequence

Quinn Lu John Bauer
Stratagene Cloning Systems, Inc.

The ESP LIC cloning and expression system features rapid and high-yield production of recombinant proteins in yeast. This system uses ligation-independent cloning (LIC) for efficient and directional cloning of PCR products immediately downstream of the glutathione-S-transferase (GST) purification tag and the recognition site for enterokinase. After expression and single-column purification of the GST fusion protein, the purification tag can be removed with enterokinase, which cleaves C-terminal to its recognition site, producing polypeptides with completely native amino acid sequences.

figure 1

Stratagenes ESP LIC cloning and expression system offers rapid and high-yield production of recombinant proteins in a eukaryotic organism. Because the system uses the yeast Schizosaccharomyces pombe as the expression host, proteins undergo many of the posttranslational modifications that are often required for their biological activity. This system also features the new pESP-2 vector (figure 1), which retains all of the unique properties of the pESP-1 vector,¹ such as tight transcriptional regulation by thiamine and high expression levels. The distinct advantage that the pESP-2 vector offers is efficient and directional LIC^2-4 cloning of PCR products immediately downstream of a GST purification tag.

Cloning PCR products using the LIC strategy is so efficient that as little as 20 ng of PCR product is required for each cloning experiment. PCR-generated sequence errors are minimized by using fewer amplification cycles (e.g., 15 cycl
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