( 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 cycles 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 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 fusion protein gave approximately 4 mg per liter (data not shown). The GST-chicken calmodulin recombinant protein produced approximately 12 mg per liter of fusion protein. Figure 3 shows the one-column purification of the GST-calmodulin fusion protein and subsequent digestion with enterokinase.

Conclusions

The pESP LIC cloning and protein purification kits allow efficient and directional cloning of high-fidelity PCR products immediately downstream of the GST purification tag and the recognition site for enterokinase. Single-step purification of the recombinant GST fusion protein and removal of the purification tag by enterokinase cleavage yield polypeptides with completely native amino acid sequences. The ESP LIC cloning and protein purification kit includes all the necessary components for LIC cloning, expression in S.pombe and GST affinity purification.

Materials and Methods

For protein induction, the expression strain was inoculated into 10 ml of YES media and incubated at 30C overnight. This culture was used to inoculate another 10 ml of YES media and was incubated at 30C until the culture reached mid-log phase. The cells were collected by centrifugation, washed once with 50 ml of sterilized water, resuspended in 10 ml of EMM media with 25 M thiamine (repressed) or without thiamine (induced) and incubated at 30C for 18 to 20 hours. Crude lysates were prepared by washing cells in PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ and 1% Triton X-100), then lysing cells by vortexing with glass beads in PBST buffer containing protease inhibitors (1 mM PMSF, 1 g/ml aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin). Soluble protein was bound to glutathione agarose, the beads were washed and the fusion protein was eluted with 50 mM reduced glutathione. Samples of cells from induced conditions are shown (Figure 3A). Of the eluted GST-calmodulin fusion protein, 20 g was cleaved in a 60-l reaction volume using Stratagenes Enterokinase (figure 3B) at room temperature for 10 or 20 hours.

Acknowledgments

We would like to thank Peter Vaillancourt, Alan Greener and Denise Wyborski for discussions and suggestions and Cherie Dewar for technical assistance.

REFERENCES

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