Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Quantitation of CP4 5-Enolpyruvylshikimate-3-Phosphate Synthase in Soybean by Two-Dimensional Gel Electrophoresis

  • KIM YEON-HEE (Department of Food Science and Technology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • CHOI SEUNG JUN (Department of Food Science and Technology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • LEE HYUN-AH (Department of Food Science and Technology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • MOON TAE WHA (Department of Food Science and Technology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University)
  • Published : 2006.01.01
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Abstract

Changes of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in the glyphosate-tolerant Roundup Ready soybean were examined using purified CP4 EPSPS produced in cloned Escherichia coli as a control. CP4 EPSPS in genetically modified soybean was detected by twodimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with databases. CP4 EPSPS in soybean products was resolved on 2-DE by first isoelectric focusing (IEF) based on its characteristic pI of 5.1, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on its molecular mass of 47.5 kDa. We quantified various percentages of soybean CP4 EPSPS. The quantitative analysis was performed using a 2D software program on artificial gels with spots varying in Gaussian volumes. These results suggested that 2-DE image analysis could be used for quantitative detection of GM soybean, unlike Western blotting.

Keywords

References

  1. Ahmed, F. E. 2002. Detection of genetically modified organisms in foods. Trends Biotech. 20: 215-223 https://doi.org/10.1016/S0167-7799(01)01920-5
  2. Choe, L. H. and K. H. Lee. 2003. Quantitative and qualitative measure of intra-laboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis. Electrophoresis 24: 3500- 3507 https://doi.org/10.1002/elps.200305614
  3. Corpillo, D., G. Gardini, A. M. Vaira, M. Basso, S. Aime, G. P. Accotto, and M. Fasano. 2004. Proteomics as a tool to improve investigations of substantial equivalence in genetically modified organisms: The case of a virus-resistant tomato. Proteomics 4: 193-200 https://doi.org/10.1002/pmic.200300540
  4. Gorg, A., O. Drews, and W. Weiss. 2003. Equilibration of IPG gel strips, pp 420-421. In R. J. Simpson (ed.), Purifying Proteins for Proteomics. Cold Spring Harbor Laboratory Press, New York, U.S.A
  5. Henzel, W. J., T. M. Billeci, J. T. Stults, S. C. Wong, C. Grimley, and C. Watanabe. 1993. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90: 5011-5015
  6. Hird, H., R. Pumphrey, P. Wilson, J. Sunderland, and P. Reece. 2000. Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis. Electrophoresis 21: 2678-2683 https://doi.org/10.1002/1522-2683(20000701)21:13<2678::AID-ELPS2678>3.0.CO;2-V
  7. Jensen O. N., M. Wilm, and M. M. Schevchenko. 1999. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels, pp. 513-530. In A. J. Link (ed.), 2-D Proteome Analysis Protocols. Humana Press, Totowa, N.J., U.S.A
  8. Kast, J., C. E. Parker, K. Drift, D. Michael, S. L. Milgram, W. Matthias, M. Howell, and C. H. Borchers. 2003. Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification. Rapid Commun. Mass Spectrom. 17: 1825- 1834 https://doi.org/10.1002/rcm.1114
  9. Kim, M. J., H. J. Chung, S. M. Park, S. G. Park, D. K. Chung, M. S. Yang, and D. H. Kim. 2004. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)- based cloning of enolase, ENO1, from Cryphenectria parasitica. J. Microbiol. Biotechnol. 14: 620-627
  10. Kwak, B. Y., B. J. Kwon, C. H. Kweon, and D. H. Shon. 2003. Detection of Fusarium species by enzyme-linked immunosorbent assay using monoclonal antibody. J. Microbiol. Biotechnol. 13: 794-799
  11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  12. Liao, X., T. Ying, H. Wang, J. Wang, Z. Shi, E. Feng, K. Wei, Y. Wang, X. Zhang, L. Huang, G. Su, and P. Huang. 2003. A two-dimensional proteome map of Shigella flexneri. Electrophoresis 24: 2864-2882 https://doi.org/10.1002/elps.200305519
  13. Mahon, P. and P. Dupree. 2001. Quantitative and reproducible two-dimensional gel analysis using Phoretix 2D Full. Electrophoresis 22: 2075-2085 https://doi.org/10.1002/1522-2683(200106)22:10<2075::AID-ELPS2075>3.0.CO;2-C
  14. O'Farrel, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007-4021
  15. Ong, S. E. and A. Pandey. 2001. An evaluation of use of two-dimensional gel electrophoresis in proteomics. Biol. Engineer. 18: 195-205
  16. Padgette, S. R., K. H. Kolacz, X. Delannay, D. B. Re, B. J. LaVallee, C. N. Tinius, W. K. Rhodes, Y. I. Otero, G. F. Barry, D. A. Eichholtz, V. M. Peschke, D. L. Nida, N. B. Taylor, and G. M. Kishore. 1995. Development, identification and characterization of a glyphosate-tolerant soybean line. Crop Sci. 35: 1451-1461 https://doi.org/10.2135/cropsci1995.0011183X003500050032x
  17. Raman, B., A. Cheung, and M. R. Marten. 2002. Quantitative comparison and evaluation of two commercially available, two-dimensional electrophoresis image analysis software packages, Z3 and Melanie. Electrophoresis 23: 2194-2202 https://doi.org/10.1002/1522-2683(200207)23:14<2194::AID-ELPS2194>3.0.CO;2-#
  18. Rogan, G. J., Y. A. Dudin, T. C. Lee, K. M. Margin, J. D. Astwood, N. S. Bhakta, S. P. R. Leach, and R. L. Fuchs. 1999. Immunodiagnostic methods for detection of 5- enolpyruvylshikimate-3-phosphate synthate in Roundup Ready$^{\circledR}$ soybeans. Food Control 10: 407-414 https://doi.org/10.1016/S0956-7135(99)00083-3
  19. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68: 850-858 https://doi.org/10.1021/ac950914h
  20. Shun-Tang, G., T. Ono, and M. Mikami. 1997. Interaction between proteins and lipids in soybean milk at elevated temperature. J. Agric. Food Chem. 45: 4601-4605 https://doi.org/10.1021/jf970417x
  21. Sickmann, A., K. Marcus, H. Schafer, E. B- Dorje, S. Lehr, A. Herkner, S. Suer, I. Bahr, and H. E. Meyer. 2001. Identification of post-translationally modified proteins in proteome studies. Electrophoresis 22: 1669-1676 https://doi.org/10.1002/1522-2683(200105)22:9<1669::AID-ELPS1669>3.0.CO;2-7
  22. Son, D. Y., J. H. Moon, M. A. Kang, D. H. Shon, S. K. Lee, H. Y. Sim, Y. S. Han, and S. I. Lee. 2004. Immunoblotting assay for glyphosate-tolerant genetically modified soybean in soybean products. Kor. J. Food Sci. Technol. 36: 369- 374
  23. Towbin, H., T. Staebelin, and J. Gordin. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nat. Acad. Sci. USA 76: 4350-4354
  24. Yamada, M., K. Murakami, J. C. Wallingford, and Y. Yuki. 2002. Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry. Electrophoresis 23: 1153-1160 https://doi.org/10.1002/1522-2683(200204)23:7/8<1153::AID-ELPS1153>3.0.CO;2-Y