Versatile eukaryotic expression and protein purification vector
Quinn Lu Tanya Hosfield
Stratagene has constructed and tested the new pESP-3 vector, which can be used for C-terminal fusion with glutathione-S-transferase (GST) and for protein production in Schizosaccharomyces pombe. After expression and purification of the GST fusion protein using the ESP yeast protein expression and purification system, the GST tag can be removed by proteolytic cleavage with thrombin or enterokinase.
Expression and purification of heterologous genes often involve fusion of the gene of interest with a purification tag at either the N-terminus or C-terminus. The Schistosoma japonicum GST gene product has been popularly used as a protein fusion tag for affinity purification of GST fusion proteins.1 Stratagenes ESP yeast protein expression and purification system includes the pESP-1 and pESP-2 vectors for N-terminal tagging with GST.2-4 The fission yeast S. pombe, which is used in the ESP system, possesses characteristics that closely resemble those of higher eukaryotic organisms regarding chromosome structure and function, cell cycle control and RNA splicing.5 Stratagenes ESP system features high-level protein production in S. pombe, thereby retaining the posttranslational modifications of eukaryotic proteins that may be critical for their structure and function.
The availability of an expression vector for C-terminal tagging with GST is desirable since the stability and activity of fusion proteins may vary with the location of the tag.6-8 In addition, by tagging a protein at the C-terminus, only fully translated proteins will have the GST affinity tag, ensuring purification of full-length fusion proteins. Stratagene has constructed the pESP-3 vector for C-terminal tagging of a protein of interest with GST. The pESP-3 vector can also be used to express proteins without a tag for in vivo analysis of gene function.
The pESP-3 vector (figure 1) is a derivative of the pESP-2 vector. Similar to the pESP-1 and pESP-2 vectors, the new pESP-3 vector contains the following features: (1) a ColE1 origin of replication and an ampicillin-resistance gene (ampr), allowing vector replication and antibiotic selection in E. coli; (2) the ars1 fragment, providing the origin of replication for the vector to replicate autonomously in S. pombe; (3) a LEU2-d gene from Saccharomyces cerevisiae, serving as a selection marker for transforming the expression vector into S. pombe cells (strain SP-Q01, leu1-32h-) and (4) the expression cassette, containing the S. pombe nmt1 promoter, a translation start site, a multiple cloning site (MCS), a GST protein-tag sequence and the nmt1 transcription termination signal. 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.9 When activated, the nmt1 promoter has been shown to be one of the strongest promoters in S. pombe.10
When a gene of interest is inserted into the MCS of the pESP-3 vector, a C-terminal fusion with the GST peptide (~27 kDa) results, which facilitates one-step purification of the GST fusion protein.1 In order to provide convenient removal of the GST tag after purification, a sequence coding for the recognition and cleavage sites for enterokinase and thrombin have been engineered between the MCS and the GST gene. Cleavage of the fusion protein with enterokinase results in a protein with the FLAG epitope tag attached on its C-terminus; cleavage by thrombin removes both the GST sequence and the FLAG epitope.
The chicken calmodulin gene was inserted into the Nde I and BamH I sites of the pESP-3 vector, serving as a test gene for protein expression and purification. The calmodulin gene primers were designed such that the calmodulin gene would be fused in frame at its C-terminus to the GST gene. Recombinant clones were identified by colony PCR analysis using vector-specific primers (the pESP-3 vector forward and reverse primers) that flank the MCS. A positive clone was chosen for further study.
Figure 2 Panel A
figures 2 Panel B
figures 2 Panel C
The pESP-3 vector containing the chicken calmodulin gene was transformed into the S. pombe strain SP-Q01 using a standard protocol.11 Figure 2 Panel A shows the expression of the calmodulin-GST fusion protein and purification by one-step chromatography using the glutathione-agarose beads1 contained in the ESP yeast protein expression and purification kit. The level of induced expression of the calmodulin-GST fusion protein is estimated to be 10% to 15% of the total soluble protein. Approximately 12.5 mg of the calmodulin-GST fusion protein was obtained from 1 liter of induced culture. After purification of the fusion protein, the GST tag was removed by incubating the fusion protein with either enterokinase or thrombin (figures 2 Panel B and Panel C). In similar experiments, the firefly luciferase gene was expressed using the pESP-3 vector. The luciferase-GST fusion protein was purified to near homogeneity with a yield of 12.5 mg/liter (data not shown).
Stratagenes FLAG Western Detection Kit can be used to monitor expression and purification processes. With this kit, recombinant fusion proteins that are tagged with the FLAG epitope at N-terminal, C-terminal and internal positions can be detected. As shown in figure 3, the FLAG epitope contained in the calmodulin-GST fusion protein can be detected before and after purification of the fusion protein.
Stratagene previously introduced the pESP-1 and pESP-2 vectors (figure 4), which feature N-terminal tagging with GST for purification.2-4 In addition, the pESP-2 vector offers the option of cloning PCR products through ligation-independent cloning (LIC).12 When the LIC method is used, polypeptides without extraneously added amino acid residues can be obtained after removing the GST tag by enterokinase cleavage.3,4 Both the pESP-1 and pESP-2 vectors are available separately and as components of the ESP system and ESP LIC systems, respectively. The ESP system has been extensively tested for inducible expression of a variety of eukaryotic proteins, including human MEK kinases and rat c-Jun N-terminal kinase (JNK). In these tests, up to 12.5 mg/liter of recombinant proteins has been obtained (table 1).
Proteins Produced in S. pombe Using the ESP System
pESP-1, pESP-2, pESP-3
pESP-1, pESP-2, pESP-3
* Yields estimated from small-scale preparations.
** MEK1CA is a constitutively active mutant of MEK1.
Stratagenes new pESP-3 yeast expression vector allows C-terminal fusions with GST for one-step purification of full-length fusion proteins. The GST tag can be removed by proteolytic cleavage with either thrombin or enterokinase. Stratagenes ESP yeast protein expression and purification system and the pESP-1, pESP-2 and pESP-3 vectors offer researchers the choice for either N-terminal or C-terminal tagging with GST. Because the eukaryotic organism, S. pombe, is used for high-level expression and one-step purification of proteins, the posttranslational modifications of eukaryotic proteins are retained.
Colony PCR analysis. After transformation, E. coli colony PCR analysis was used to verify insertions in the pESP-3 vector using primers flanking the MCS of the pESP-3 vector. The forward primer, 5-GGCATATCATCAATTGAATAAG-3, corresponds to nucleotide sequence 6968-6989. The reverse primer, 5-GATATTCCAAAAGAAGTCGAGTGGG-3, corresponds to nucleotide sequence 7235-7259. These primers can also be used to confirm the cloning junctions by sequencing.
Expression and purification of recombinant protein. For protein induction, the expression strain (SP-Q01 transformed with the pESP-3 vector containing the calmodulin gene) was inoculated into 5 ml of YES media and incubated at 30C overnight. A portion of the overnight culture was used to inoculate 10 ml of YES media (OD600=0.1). Four to five hours of further incubation at 30C were required for the culture to reach mid-logarithmic phase (OD600=0.5). The cells were collected by centrifugation at 1000 x g in a benchtop centrifuge and washed once with 50 ml of sterilized water. The cells were then resuspended into 10 ml of EMM media with 25 M thiamine (repressed) or without thiamine (induced) and grown at 30C for 18 to 20 hours. The cells were collected and washed once with PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 1% Triton X-100), and the cell pellet was further resuspended in 500 l of PBST buffer containing protease inhibitors (1 mM PMSF, 1 g/ml aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin). Five hundred micrograms of acid-washed glass beads (0.4- to 0.6-mm diameter) were added, and the cells were lysed by vortexing at 4C for 5 minutes. After centrifugation in a microcentrifuge for 5 minutes at 12,000 x g, the supernatant (crude lysate) was saved and stored at -70C for further purification and analyses. The GST-tagged fusion protein was purified as previously described.1
We would like to thank Wei-Ping Yang, Connie Hansen, Edward Aranas and members of the Stratagene Genetic Systems group for discussions and suggestions.
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