Navigation Links
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.


'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Epitope-Tagging Vectors for Functional Analysis in Yeast
2. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
3. Electroporation of Yeast Artificial Chromosomes
4. New Automated Yeast Cell Counter Ends Tedium & Errors of Manual Counting
5. High Yield Protein Production from Pichia pastoris Yeast: A Protocol for Benchtop Fermentation
6. A New C-Terminal GST Vector for Protein Production in S. pombe
7. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
8. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
9. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
10. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
11. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
Post Your Comments:
(Date:8/29/2014)... (PRWEB) August 29, 2014 Intrinsic ... 13485:2003 certified, GAMP® 5 compliant imaging core lab, ... international Phase II clinical trial to assess a ... Throughout this trial, Intrinsic Imaging will provide comprehensive ... to, protocol and charter development, site qualification, site ...
(Date:8/28/2014)... Ca. (PRWEB) August 28, 2014 Best ... rating for an alcohol-based hand sanitizer, asks food processors ... by comparing the hand sanitizer they’re currently using to ... . Hand hygiene is critical to fighting cross-contamination and ... Best Sanitizers believes there are key criteria that ...
(Date:8/28/2014)... an electronic which is not only based on ... spin and the spin-related magnetism. Spin-charge converters enable ... vice versa. Recently, the research group of Professor ... Johannes Gutenberg University Mainz in collaboration with researchers ... the first time realised a new, efficient spin-charge ...
(Date:8/28/2014)... 28, 2014  Next month, executives from clinical trial marketing ... events beginning with Patient-Centered Clinical Trials 2014 , to ... Boston , September 4-5. Patient recruitment experts Bonnie ... Fleishman will share insights on the benefits of employing ... tactics – from media to mobile apps – can be ...
Breaking Biology Technology:Intrinsic Imaging Awarded Phase II Clinical Trial to Assess New Treatment for Non-Hodgkin’s Lymphoma 2Food Processors and Food Handlers are Encouraged to Prepare for Fall Harvest by Evaluating Their Current Hand Sanitizer Using Nine Key Criteria 2Food Processors and Food Handlers are Encouraged to Prepare for Fall Harvest by Evaluating Their Current Hand Sanitizer Using Nine Key Criteria 3A new, tunable device for spintronics 2A new, tunable device for spintronics 3BBK Worldwide Leads Sessions at Key September Events 2
... RALEIGH, N.C. , July 30 SmartGene, Inc., ... genetic data, today announced that it has entered into an agreement ... and integrated Web-based services to support more rapid and precise identification ... Some bacteria and fungi do not grow quickly or ...
... , , SAN ... CXM ) today announced plans to develop a DNA-based orthobiologics ... Repair Company that will initially focus on non-union bone fractures ... disc disease. Orthobiologics is a rapidly growing segment of ...
... , ALPHARETTA, Ga., July 30 Brookstone Pharmaceuticals, ... Philip Vogt as Executive Vice President, Sales and Marketing. , ... of pharmaceutical experience and will lead the sales and ... served as President of Ethex Corporation in St. Louis, MO. ...
Cached Biology Technology:SmartGene's Services for Faster, More Precise Identification of Bacteria and Fungi to be Used by The Johns Hopkins Hospital 2Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 2Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 3Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 4Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 5Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 6Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 7Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 8Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 9Cardium Announces New Orthobiologics Product Initiative Extending Gene Activated Matrix Technology to Deliver Bone Growth Factors 10
(Date:8/29/2014)... climatic shifts over the last 120,000 years may ... team of researchers led by City College of ... new biodiversity metric called "phylogeographic endemism." , It ... within species is restricted in geographical space. , ... 14 other researchers from institutions in Brazil, Australia ...
(Date:8/29/2014)... in German . ... natural nitrogen cycle on Earth and in biological wastewater ... to depend on nitrite as their source of energy. ... a microbiologist at the University of Vienna, has now ... alternative source of energy. The oxidation of hydrogen with ...
(Date:8/29/2014)... of marine phytoplankton, such as the prolific bloomer ... (thiamine), researchers have discovered. The finding contradicts the common ... eukaryotic microbes depend on scarce supplies of thiamine in ... way to think about the ocean," says CIFAR Senior ... ISME Journal paper with CIFAR fellows John ...
Breaking Biology News(10 mins):CCNY team defines new biodiversity metric 2Hydrogen powers important nitrogen-transforming bacteria 2Hydrogen powers important nitrogen-transforming bacteria 3Not all phytoplankton in the ocean need to take their vitamins 2Not all phytoplankton in the ocean need to take their vitamins 3
... to combat metastatic cancer Cancer cells spread and ... Stimulation of the immune system can help to eliminate cancer ... system to ignore cancer cells. Regulatory T cells are immune ... this issue of the Journal of Clinical Investigation , ...
... the world today issued a stark warning that, without major ... the majority of the 9 billion people on Earth will ... water, an absolutely essential natural resource for which there is ... believe, entirely avoidable." The scientists bluntly pointed to chronic ...
... Rollins School of Public Health at Emory University, along ... received a $4 million grant over four years to ... Exposome Research Center: Understanding Lifetime Exposures). The grant is ... States. The HERCULES Center is funded by ...
Cached Biology News:JCI early table of contents for May 24, 2013 2JCI early table of contents for May 24, 2013 3JCI early table of contents for May 24, 2013 4JCI early table of contents for May 24, 2013 5JCI early table of contents for May 24, 2013 6JCI early table of contents for May 24, 2013 7JCI early table of contents for May 24, 2013 8A majority on Earth face severe self-inflicted water woes within 2 generations: Scientists 2A majority on Earth face severe self-inflicted water woes within 2 generations: Scientists 3A majority on Earth face severe self-inflicted water woes within 2 generations: Scientists 4Emory, Georgia Tech receive first human exposome center grant in US 2Emory, Georgia Tech receive first human exposome center grant in US 3
... is a magnetic bead-based PCR ... sample transfer, centrifugation or filtration ... and large PCR extension products ... Agencourt AMPure can easily be ...
... 1.5L PYREX trypsinizing flasks are ... samples into cell suspensions by ... beaded neck accepts cotton plugs. ... agitation. • Height of Baffles ...
... for stabilizing 50g of purified RNA samples for ... delivers all of the advantages and applications you ... the RNA can be used for enzymatic applications ... has been optimised for the stabilization of aqueous ...
... culture flasks have triple baffles located at ... achieve maximal oxygen transfer to culture medium. ... A wide range of optional caps are ... colored caps for ease of sorting and ...
Biology Products: