Navigation Links
A New C-Terminal GST Vector for Protein Production in S. pombe


Versatile eukaryotic expression and protein purification vector

Quinn Lu Tanya Hosfield
Stratagene

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

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.

Expression and Purification of Chicken Calmodulin

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.

figure 3

Other pESP Vectors

figure 4

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).

Table 1
Proteins Produced in S. pombe Using the ESP System

Genes

Sizes
(kDa)

Yields*
(mg/liter)

Vectors

Note

GST (vector)

27

12.5

pESP-1, pESP-2, pESP-3

GST-Calmodulin

43

12.5

pESP-1, pESP-2, pESP-3

GST-JNK

74

5

pESP-1

GST-PHAS I

41

5

pESP-1

GST-MEK1

69

4

pESP-1, pESP-2

GST-MEK1CA

66

4

pESP-1, pESP-2

**

GST-Thioredoxin

39

8

pESP-1

GST-CAT

51

8

pESP-1

GST-Pfu

120

1

pESP-1

GST-Krs2

84

2

pESP-2

GST-Elk1

72

<1

pESP-2

GST-Luciferase

64

12.5

pESP-3

GST-Cre

55

<1

pESP-2

* Yields estimated from small-scale preparations.
** MEK1CA is a constitutively active mutant of MEK1.

Conclusions

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.

Methods

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

Acknowledgments

We would like to thank Wei-Ping Yang, Connie Hansen, Edward Aranas and members of the Stratagene Genetic Systems group for discussions and suggestions.

REFERENCES
  1. Smith, D.B., and Johnson, K.S. (1988) Gene 67: 31-40.

  2. Lu, Q., Jerpseth, B., Sanchez, T., Bauer, J.C., and Greener, A. (1997) Strategies 10: 4-6.

  3. Lu, Q., Bauer, J.C., and Greener, A. (1997) Gene In press.

  4. Lu, Q., and Bauer, J.C. (1997). Strategies 10: 72-74.

  5. Sipiczki, M. (1989) In Molecular Biology of Fission Yeast (A. Nisim, P. Young, and B.F. Johnson, eds), pp. 431-452. Academic Press, San Diego, California.

  6. La Vallie, E.R., and McCoy, J.M. (1995) Curr. Opin. Biotechnol. 6: 501-506.

  7. Sharrocks, A.D. (1994) Gene 138: 105-108.

  8. Williams, G., et al. (1995) Biochemistry 34: 1787-1797.

  9. Maundrell, K. (1990) J. Biol. Chem. 265: 10857-10864.

  10. Forsburg, S. (1993) Nucleic Acids Res. 21: 2955-2956.

  11. Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194: 795-823.

  12. Aslanidis, C., and de Jong, P.J. (1990) Nucleic Acids Res. 18: 6069-6074.


'"/>

Source:


Page: All 1 2 3 4 5 6 7 8 9

Related biology technology :

1. Generate Adenovirus Vectors in E. coli by Homologous Recombination with the AdEasy Adenoviral Vector System
2. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
3. Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
4. Epitope-Tagging Vectors for Functional Analysis in Yeast
5. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
6. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
7. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
8. A New Lambda Vector for Mammalian Expression
9. Versatile Reporter Vectors for Monitoring Viral Transduction
10. High-Titer Retroviral Vectors for Gene Delivery
11. Improved Vectors for PathDetect Trans-Reporting Systems
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: New Terminal GST Vector for Protein Production pombe

(Date:1/15/2014)... Hills, IL (PRWEB) January 15, 2014 ... to find hard-working items for the lab, from fluid ... as Guaranteed-in-Stock (GIS)—ready to ship when you order. ... Masterflex® Peristaltic Pumps , from the L/S® model for ...
(Date:1/15/2014)... January 15, 2014 A study has been ... the Formula 1 track could help to tackle the problem ... (MAT), Stowhealth (a GP surgery based in Stowmarket) and academics ... Simplyhealth. Telemetry technology, which is inspired by equipment ...
(Date:1/15/2014)... 2013 was a banner year of ... They saw continued independent research led by the team ... a $1 million grant from the Susanne Marcus Collins ... a peer reviewed journal, Amy Grant highlighted Brainwave Optimization® ...
(Date:1/14/2014)... Carahsoft and CDS Federal Services have scheduled a ... EST (11am PST), “Natural Language Processing: Converting Raw Data ... can turn raw, heterogeneous data into actionable knowledge to ... webinar will last approximately one hour. , Synopsis: Big ...
Breaking Biology Technology:Cole-Parmer Begins 2014 with the Release of Preferred Solutions 2Formula 1 Technology Tackles Obesity in Unique Healthcare Partnership 2Formula 1 Technology Tackles Obesity in Unique Healthcare Partnership 3Formula 1 Technology Tackles Obesity in Unique Healthcare Partnership 4Dynamic Innovative Technology Showcased at Scottsdale Company’s Open House 2Dynamic Innovative Technology Showcased at Scottsdale Company’s Open House 3Webcast - Natural Language Processing: Converting Raw Data into Actionable Knowledge – Hosted by Carahsoft and CDS Federal Services 2
... /PRNewswire-FirstCall/ - The Board of Directors of SemBioSys ... pharmaceuticals in crop plants, today announced that current ... to the new position of President and CEO ... James Szarko, currently the corporation,s Chief Financial Officer, ...
... Pharmaceuticals, a pioneer in the development of novel products ... disease, announced that Ted Hibben has joined the company ... from Coley Pharmaceutical Group, where he was most recently ... played a key role in the company,s $233 million ...
... NEW YORK, Jan. 9 /PRNewswire-Asia-FirstCall/ -- American Oriental,Bioengineering, ... "the Company"), a leading marketer,distributor and manufacturer of ... its recent purchase of buildings and land in ... , Mr. Tony Liu, ...
Cached Biology Technology:SemBioSys announces senior management changes 2SemBioSys announces senior management changes 3SemBioSys announces senior management changes 4Cequent Names Ted Hibben Chief Business Officer 2Cequent Names Ted Hibben Chief Business Officer 3American Oriental Bioengineering Comments on Recent Property Purchase 2American Oriental Bioengineering Comments on Recent Property Purchase 3
(Date:4/23/2014)... a technique used to read quickly. It involves ... non-essential words and/ or sentences. Similarly to humans, ... quickly "read" genetic information. Genes that need to ... smaller the encoding message, the easier it will ... Instituto Gulbenkian de Cincia (IGC, Portugal) and Centre ...
(Date:4/23/2014)... Los Angeles, London (April 23, 2014). "I think one can ... energy pathwayas well as doing everything else that we canthen ... says Tom Wigley, one of the world,s foremost climate researchers, ... Scientists , published by SAGE. Refusing to take significant action ... will eventually be needed to address the problem, Wigley explains ...
(Date:4/22/2014)... are all sorts of signaling strategies in nature. Peacocks ... satin bowbirds build specialized stick structures, called bowers, and ... bitterling males show off bright nuptial coloration during spawning ... communicate with others. , ... fitness implications for individuals that are either signaling or ...
Breaking Biology News(10 mins):Cell division speed influences gene architecture 2Is nuclear power the only way to avoid geoengineering? 2Best practices in communication for the animal world 2Best practices in communication for the animal world 3
... Cambridge, MA --- The McGovern Institute for Brain Research ... Scolnick Prize in Neuroscience to Jeremy Nathans, a Howard ... and genetics, neuroscience, and opthalmology at Johns Hopkins University ... the Scolnick Prize lecture entitled "The Evolution of Trichromatic ...
... 2009) The Wildlife Conservation Society announced today a ... are unusually resilient to climate change due to improved ... WCS announced the results of the study at the ... week in Phuket, Thailand. The study, published in ...
... release is available in <A HREF=",http://chinese.eurekalert.org/zh/emb_releases/2009-04/sfeb-roc042209.php,">Chinese . ... State University College of Medicine, Hershey, Pennsylvania ... Opioid Growth Factor (OGF, [Met5]-enkephalin), a clinically ... translocation and reliant on the integrity of ...
Cached Biology News:MIT: Jeremy Nathans to deliver Scolnick Prize lecture 2'Super reefs' fend off climate change, study says 2Regulation of cell proliferation by the OGF-OGFr axis is dependent on nuclear localization signals 2Regulation of cell proliferation by the OGF-OGFr axis is dependent on nuclear localization signals 3
... of Cell Cultures (ECACC) in conjunction with ... West of England, Bristol, UK, have produced ... of HER2, oestrogen and progresterone receptor assays. ... of formalin fixed, breast cancer cell lines; ...
... the highly potent TURBO DNase (patent pending) in ... set in DNA removal capabilities. TURBO DNase is ... is much more efficient than wild type DNase ... DNA. TURBO DNase binds DNA substrates 6-fold more ...
Lyophilized. Purity: ≥90% by electrophoresis. Reconstitute in 0.3% NaCl. Prepared from serum that has been shown by certified tests to be negative for HBsAg and for HIV and HCV antibodies. ...
PP2B-Abeta (C-20)...
Biology Products: