Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Enhanced Proteomic Analysis of Streptomyces peucetius Cytosolic Protein Using Optimized Protein Solubilization Protocol

  • Lee, Kwang-Won (Interdisciplinary Program for Biochemical Engineering and Biotechnology, and Institute of Molecular Biology and Genertics) ;
  • Song, Eun-Jung (School of Chemical & Biological Engineering, Seoul National University) ;
  • Kim, June-Hyung (School of Chemical & Biological Engineering, Seoul National University) ;
  • Lee, Hei-Chan (Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University) ;
  • Liou, Kwang-Kyoung (Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University) ;
  • Sohng, Jae-Kyung (Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University) ;
  • Kim, Byung-Gee (Interdisciplinary Program for Biochemical Engineering and Biotechnology, and Institute of Molecular Biology and Genertics)
  • Published : 2007.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Buttner, K., J. Bernhardt, C. Scharf, R. Schmid, U. Mader, C. Eymann, H. Antelmann, A. Volker, U. Volker, and M. Hecker. 2001. A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 22: 2908-2935 https://doi.org/10.1002/1522-2683(200108)22:14<2908::AID-ELPS2908>3.0.CO;2-M
  3. Chinnasamy, G. and C. Rampitsch. 2006. Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds. Biochim. Biophys. Acta 1764: 641-644 https://doi.org/10.1016/j.bbapap.2005.10.003
  4. Dekleva, M. L., J. A. Titus, and W. R. Strohl. 1985. Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium. Can. J. Microbiol. 31: 287-294 https://doi.org/10.1139/m85-053
  5. Eymann, C., A. Dreisbach, D. Albrecht, J. Bernhardt, D. Becher, S. Gentner, T. Tam le, K. Buttner, G. Buurman, C. Scharf, S. Venz, U. Volker, and M. Hecker. 2004. A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics 4: 2849-2876 https://doi.org/10.1002/pmic.200400907
  6. Fountoulakis, M. and B. Takacs. 2001. Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels. Electrophoresis 22: 1593-1602 https://doi.org/10.1002/1522-2683(200105)22:9<1593::AID-ELPS1593>3.0.CO;2-6
  7. Gatlin, C. L., G. R. Kleemann, L. G. Hays, A. J. Link, and J. R. Yates 3rd. 1998. Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 263: 93-101 https://doi.org/10.1006/abio.1998.2809
  8. Giometti, C. S., C. Reich, S. Tollaksen, G. Babnigg, H. Lim, W. Zhu, J. Yates, and G. Olsen. 2002. Global analysis of a 'simple' proteome: Methanococcus jannaschii. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782: 227-243 https://doi.org/10.1016/S1570-0232(02)00568-8
  9. Gorg, A., W. Weiss, and M. J. Dunn. 2004. Current twodimensional electrophoresis technology for proteomics. Proteomics 4: 3665-3685 https://doi.org/10.1002/pmic.200401031
  10. Herbert, B. R., M. P. Molloy, A. A. Gooley, B. J. Walsh, W. G. Bryson, and K. L. Williams. 1998. Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19: 845-851 https://doi.org/10.1002/elps.1150190540
  11. Hesketh, A. R., G. Chandra, A. D. Shaw, J. J. Rowland, D. B. Kell, M. J. Bibb, and K. F. Chater. 2002. Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor. Mol. Microbiol. 46: 917-932 https://doi.org/10.1046/j.1365-2958.2002.03219.x
  12. Hoving, S., B. Gerrits, H. Voshol, D. Muller, R. C. Roberts, and J. van Oostrum. 2002. Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2: 127-134 https://doi.org/10.1002/1615-9861(200202)2:2<127::AID-PROT127>3.0.CO;2-Y
  13. Kieser, T. 2000. Practical Streptomyces Genetics. John Innes Foundation, Norwich
  14. Kim, Y. H., K. Y. Han, K. Lee, and J. Lee. 2005. Proteome response of Escherichia coli fed-batch culture to temperature downshift. Appl. Microbiol. Biotechnol. 68: 786-793 https://doi.org/10.1007/s00253-005-0053-3
  15. Lanne, B., F. Potthast, A. Hoglund, H. Brockenhuus von Lowenhielm, A. C. Nystrom, F. Nilsson, and B. Dahllof. 2001. Thiourea enhances mapping of the proteome from murine white adipose tissue. Proteomics 1: 819-828 https://doi.org/10.1002/1615-9861(200107)1:7<819::AID-PROT819>3.0.CO;2-V
  16. Lee, K. W., H. S. Joo, Y. H. Yang, E. J. Song, and B. G. Kim. 2006. Proteomics for Streptomyces: 'Industrial proteomics' for antibiotics. J. Microbiol. Biotechnol. 16: 331-348
  17. Lieu, H. Y., H. S. Song, Y. N. Yang, J. H. Kim, H. J. Kim, Y. D. Park, C. S. Park, and H. Y. Kim. 2006. Identification of proteins affected by iron in Saccharomyces cerevisiae using proteome analysis. J. Microbiol. Biotechnol. 16: 946-951
  18. Lim, H., J. Eng, J. R. Yates 3rd, S. L. Tollaksen, C. S. Giometti, J. F. Holden, M. W. Adams, C. I. Reich, G. J. Olsen, and L. G. Hays. 2003. Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry. J. Am. Soc. Mass. Spectrom. 14: 957-970 https://doi.org/10.1016/S1044-0305(03)00144-2
  19. Marshall, T. and K. M. Williams. 1984. Artifacts associated with 2-mercaptoethanol upon high resolution two-dimensional electrophoresis. Anal. Biochem. 139: 502-505 https://doi.org/10.1016/0003-2697(84)90041-1
  20. Mechin, V., L. Consoli, M. Le Guilloux, and C. Damerval. 2003. An efficient solubilization buffer for plant proteins focused in immobilized pH gradients. Proteomics 3: 1299- 1302 https://doi.org/10.1002/pmic.200300450
  21. Mortz, E., T. N. Krogh, H. Vorum, and A. Gorg. 2001. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ ionization-time of flight analysis. Proteomics 1: 1359-1363 https://doi.org/10.1002/1615-9861(200111)1:11<1359::AID-PROT1359>3.0.CO;2-Q
  22. Musante, L., G. Candiano, and G. M. Ghiggeri. 1998. Resolution of fibronectin and other uncharacterized proteins by two-dimensional polyacrylamide electrophoresis with thiourea. J. Chromatogr. B Biomed. Sci. Appl. 705: 351- 356 https://doi.org/10.1016/S0378-4347(97)00545-8
  23. Nandakumar, R., M. P. Nandakumar, M. R. Marten, and J. M. Ross. 2005. Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus. J. Proteome Res. 4: 250-257 https://doi.org/10.1021/pr049866k
  24. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007-4021
  25. Okamoto, S., A. Lezhava, T. Hosaka, Y. Okamoto-Hosoya, and K. Ochi. 2003. Enhanced expression of Sadenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J. Bacteriol. 185: 601-609 https://doi.org/10.1128/JB.185.2.601-609.2003
  26. Park, H. S., S. K. Shin, Y. Y. Yang, H. J. Kwon, and J. W. Suh. 2005. Accumulation of S-adenosylmethionine induced oligopeptide transporters including BldK to regulate differentiation events in Streptomyces coelicolor M145. FEMS Microbiol. Lett. 249: 199-206 https://doi.org/10.1016/j.femsle.2005.05.047
  27. Perdewa, G. H., H. W. Schaupb, and D. P. Selivonchick. 1983. The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes. Anal. Biochem. 135: 453-455 https://doi.org/10.1016/0003-2697(83)90711-X
  28. Rabilloud, T., C. Adessi, A. Giraudel, and J. Lunardi. 1997. Improvement of the solubilization of proteins in twodimensional electrophoresis with immobilized pH gradients. Electrophoresis 18: 307-316 https://doi.org/10.1002/elps.1150180303
  29. Stanley, B. A., I. Neverova, H. A. Brown, and J. E. Van Eyk. 2003. Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium. Proteomics 3: 815-820 https://doi.org/10.1002/pmic.200300388
  30. Sutton, C. W., K. S. Pemberton, J. S. Cottrell, J. M. Corbett, C. H. Wheeler, M. J. Dunn, and D. J. Pappin. 1995. Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting. Electrophoresis 16: 308-316 https://doi.org/10.1002/elps.1150160151
  31. Tasheva, B. and G. Dessev. 1983. Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2- mercaptoethanol. Anal. Biochem. 129: 98-102 https://doi.org/10.1016/0003-2697(83)90057-X
  32. Valcu, C. M. and K. Schlink. 2006. Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples. Proteomics 6: 1599-1605 https://doi.org/10.1002/pmic.200500314
  33. Vohradsky, J., X. M. Li, G. Dale, M. Folcher, L. Nguyen, P. H. Viollier, and C. J. Thompson. 2000. Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns. J. Bacteriol. 182: 4979-4986 https://doi.org/10.1128/JB.182.17.4979-4986.2000
  34. Westermeier, R. and T. Naven. 2002. Proteomics in Practice. Wiley-VCH, Weinheim