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Yeast Protein Production System Features High Yields and One-Step,,,Purification

The ESP system provides high-yield protein production in a eukaryotic organism

Quinn Lu * Bruce Jerpseth * Tim Sanchez * John C. Bauer * Alan Greener
Stratagene Cloning Systems, Inc.

Stratagene's EPS yeast protein expression and purification systemllll provides novel opportunities for rapid, inexpensive and high-yield production of proteins in a eukaryotic organism. This system retains many eukaryotic posttranslational modifications of proteins that can be critical for the biological activity of expressed proteins. By using the yeast Schizosaccharomyces pombe as the host and glutathione-S-transferase (GST) as the protein purification tag, the ESP system expresses a protein of interest in yeast as a fusion protein with GST. The fusion protein is then purified using glutathione-agarose beads, and the GST tag can be removed from the protein by proteolytic cleavage with either bovine enterokinase or thrombin.

As numerous genes that are involved in transcriptional regulation and developmental control have been cloned and identified, the demand for homogeneously purified proteins for structural and functional analyses has increased. Although this demand can be partially fulfilled by prokaryotic expression systems, the analysis of eukaryotic proteins, whose functions are determined or influenced by posttranslational modifications, would require expression of the protein in a eukaryotic organism. Stratagene's ESP yeast protein expression and purification system has been created to meet this demand.

The yeast S. pombe is a unicellular eukaryotic organism, with properties that closely resemble higher eukaryotic organisms regarding chromosome structure and function, cell cycle control and RNA splicing.1 Stratagene's ESP system contains a cloning vector with both yeast and Escherichia coli replication origins, competent cells of the S. pombe host strain and reagents for protein induction and purification.

The pESP-1 Expression Vector

figure 1

Designed for expression in S. pombe, the pESP-1 vector (figure 1) is composed of the following parts: (1) a ColE1 origin of replication and an ampicillin resistance (Ampr) fragment, which allow vector replication and antibiotic selection in E. coli; (2) the ars1 fragment, which provides an origin of replication for the vector to replicate autonomously in S. pombe; (3) a LEU2-d gene from Saccharomyces cerevisiae, which serves as a selection marker for transforming the expression vector into S. pombe cells (strain SPQ01) and (4) the expression cassette, which contains the S. pombe nmt1 promoter, a translational start site, a GST protein-tag sequence, a multiple cloning site and the nmt1 transcription termination signal. Insertion of the gene of interest into the multiple cloning site results in an N-terminal fusion with the GST peptide, which facilitates one-step purification of the GST fusion protein.2

Figure 3

The nmt1 promoter of S. pombe is tightly repressed in the presence of thiamine (vitamin B1) in the growth medium and is highly activated upon its removal.3 When activated, the nmt1 promoter has been shown to be one of the strongest promoters in S. pombe.4 For this reason, the nmt1 promoter has been widely used to study gene function in S. pombe. Figure 3 shows Western blot analysis of the GST-calmodulin fusion protein in the pESP-1 vector that has been expressed in cells grown under repressed and induced conditions. The fusion protein cannot be detected in cells when the promoter is repressed and is abundant in cells when the promoter is induced. In addition to inducing expression of the gene product of interest, constitutive high-level expression is possible by growing the clones in media without thiamine.

The pESP-1 vector also contains DNA sequences coding for amino acid residues that are specifically recognized and cleaved by enterokinase and thrombin. These sites are located between the GST tag and the multiple cloning site (figure 1). Insertion into the multiple cloning site results in fusion proteins that can be detached from the GST tag with either protease. Cleavage by thrombin results in a fusion protein having the FLAG peptide (DYKDDDDK)5 at its N terminus; antibodies against the FLAG peptide are available from Stratagene.

Establishing the Expression Strain

The gene of interest is inserted into the multiple cloning site of the pESP-1 vector and is fused in frame to the upstream GST gene. Successful cloning can be confirmed by sequence analysis. To establish the expression strain, the verified clone is then transformed into the competent cells of S. pombe host strain SP-Q01 ((leu1-32h ) that are included in the kit. These competent cells provide optimal transformation conditions. Transformants can be identified by their ability to grow on minimal medium (EMM) agar plates supplemented with 25 M thiamine. The expression strain can be stored at 70C and used for multiple experiments.

Induction and Purification of GST-Tagged Fusion Proteins

To induce expression of the protein of interest, the S. pombe expression strain is first grown to mid-logarithmic phase in the vegetative growth medium (YES medium), which provides sufficient thiamine to repress the nmt1 promoter. Expression is induced by harvesting the cells and growing them in EMM broth without thiamine for approximately 18 hours.

Figure 2

The chicken calmodulin gene was inserted into the pESP-1 vector and expressed in S. pombe. Figure 2 shows the cell lysate samples derived from repressed and induced cells containing chicken calmodulin. When cells were grown under induced conditions, the crude lysate shows a dominant band at the size expected for a GST-calmodulin fusion protein (43 kDa) (figure2, lane 4). In the lysate of cells grown under repressed conditions, this band was absent. (Figure 2, lane 1) When the samples were subjected to one-step purification of the GST fusion protein by using GST affinity resin, the induced GST-calmodulin fusion protein was depleted in the glutathione resin flowthrough sample (Figure 2, lane 5) and was eluted to near homogeneity in the glutathione elution fraction (Figure 2, lane 6). No detectable GST fusion protein was observed when lysate derived from repressed cells was subjected to the same purification procedure (Figure 2, lane 3). Figure 3 shows Western blot analysis of the GST-calmodulin fusion protein in cell lysates derived from repressed and induced cells. The fusion protein was not detected in cells when the promoter was repressed but was abundant in cells when the promoter was induced. We have also observed that constitutive, high-level expression of GST fusion proteins is possible by inoculating the cells directly into the induction medium (EMM), which significantly simplifies the overall procedure. However, proteins potentially toxic to the host cannot be overexpressed under constitutive expression conditions. Using the S. pombe expression system, we have purified 10 different proteins, including the rat c-Jun N-terminal kinase (JNK) and the human MEK1 gene products (figure 4). Yield for these proteins has varied from 1.0 mg per liter to 12.5 mg per liter. Some of these proteins, especially MEK1, had been difficult to produce in significant quantities in E. coli.

figure 4

Removing the GST Tag by Proteolytic Cleavage with Enterokinase or Thrombin

In many cases, GST tags do not interfere with the function of the proteins. Our functional assays indicated that the biological activities of the GST-calmodulin fusion protein and the GST-MEK1 fusion protein expressed in S. pombe were retained (data not shown). However, if desired, the GST tag can be removed by treating the purified fusion protein with either enterokinase or thrombin to cleave between the GST tag and the protein of interest (figure 1). Figure 5 shows an example of protease cleavage of a GST-MEK1 fusion protein. The GST tag was efficiently detached from the MEK1 moiety by digesting the purified GST-MEK1 fusion protein with enterokinase. The enterokinase site is often preferred for cleavage since the resulting product has fewer additional amino acids remaining at the N terminus. Alternatively, if the recombinant protein is cleaved with thrombin, the resulting polypeptide will contain the FLAG peptide at its N terminus, which could facilitate further analysis of the protein.

Figure 5

Conclusions

Stratagene's new ESP yeast protein expression and purification system provides protein production in a eukaryotic organism, retaining many posttranslational modifications of recombinant proteins. The main advantage of the ESP system over other eukaryotic expression systems is quick, easy expression and purification of recombinant proteins. The S. pombe system offers highyield production with options for either inducible or constitutive expression. The kit includes easytotransform E. coli and yeast competent cells, glutathione agarose beads for protein purification, yeast media and timesaving, wellcharacterized procedures.

Acknowledgments

We thank Paul Russell and Janet Letherwood of the Research Institute of Scripps Clinic for discussions and suggestions; Joe Sorge, Peter Vaillancourt, ChaoFeng Zheng, Rebecca Mullinax, Kerstien Padgett and other members of the research and development department at Stratagene for assistance and suggestions; Bryan Macilko and Allison Fowler for media preparations.

REFERENCES

  1. Sipiczki, M. (1989) In Molecular Biology of Fission Yeast (A. Nisim,P. Young, and B.F. Johnson, eds). Academic Press, San Diego, Calif.

  2. Smith, D.B., and Johnson, K.S. (1988) Gene 67: 3140.

  3. Maundrell, K. (1990) J. Biol. Chem. 265: 1085710864.

  4. Forsburg, S. (1993) Nucleic Acids Res. 21: 29552956.

  5. Hopp, H.P., et al. (1988) Biotechnology 6: 12041210.


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