Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems

详细信息    查看全文

• 作者：Zhaopeng Li (12)
  Manfred Nimtz (1)
  Ursula Rinas (12) Ursula.Rinas@helmholtz-hzi.de
• 关键词：Escherichia coli – ; Recombinant protein production – ; Stable heavy isotope labeling – ; Selenomethionine labeling – ; Autoinduction – ; Defined medium
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2011
• 出版时间：November 2011
• 年：2011
• 卷：92
• 期：4
• 页码：823-833
• 全文大小：1.3 MB
• 参考文献：1. Bollag DM, Rozycki MD, Edelstein SJ (1996) Protein methods, 2nd edn. Wiley-Liss, Inc., New York, pp 107–151
  2. Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333. doi:
  3. Culebras JM, Moore FD (1977) Total body water and the exchangeable hydrogen. I. Theoretical calculation of nonaqueous exchangeable hydrogen in man. Am J Physiol 232:R54–R59
  4. Doublie S (1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol 276:523–530. doi:
  5. Fiaux J, Bertelsen EB, Horwich AL, Wuthrich K (2004) Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background. J Biomol NMR 29:289–297. doi:
  6. Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406. doi:
  7. Hendrickson WA (1991) Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254:51–58. doi:
  8. Hendrickson WA, Horton JR, LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J 9:1665–1672
  9. Hoffmann F, van den Heuvel J, Zidek N, Rinas U (2004) Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Technol 34:235–241. doi:
  10. Jansson M, Li YC, Jendeberg L, Anderson S, Montelione BT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7:131–141. doi:
  11. Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer WD (1995) Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol 39:59–65. doi:
  12. Leiting B, Marsilio F, O'Connell JF (1998) Predictable deuteration of recombinant proteins expressed in Escherichia coli. Anal Biochem 265:351–355. doi:
  13. Li Z, Kessler W, van den Heuvel J, Rinas U (2011) Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. Appl Microbiol Biotechnol 91:1203–1213. doi:
  14. Lu X, Sun J, Nimtz M, Wissing J, Zeng AP, Rinas U (2010) The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate. Microb Cell Fact 9:23. doi:
  15. Mac TT, Beyermann M, Pires JR, Schmieder P, Oschkinat H (2006) High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies. Protein Expr Purif 48:253–260. doi:
  16. Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75. doi:
  17. Paliy O, Bloor D, Brockwell D, Gilbert P, Barber J (2003) Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium. J Appl Microbiol 94:580–586. doi:
  18. Romanuka J, van den Bulke H, Kaptein R, Boelens R, Folkers GE (2009) Novel strategies to overcome expression problems encountered with toxic proteins: application to the production of Lac repressor proteins for NMR studies. Protein Expr Purif 67:104–112. doi:
  19. Ross A, Kessler W, Krumme D, Menge U, Wissing J, van den Heuvel J, Flohe L (2004) Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis. J Biotechnol 108:31–39. doi:
  20. Seo ES, Vargues T, Clarke DJ, Uhrin D, Campopiano DJ (2009) Preparation of isotopically labelled recombinant β-defensin for NMR studies. Protein Expr Purif 65:179–184. doi:
  21. Sreenath HK, Bingman CA, Buchan BW, Seder KD, Burns BT, Geetha HV, Jeon WB, Vojtik FC, Aceti DJ, Frederick RO, Phillips GN Jr, Fox BG (2005) Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium. Protein Expr Purif 40:256–267. doi:
  22. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234. doi:
  23. Studts JM, Fox BG (1999) Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins. Protein Expr Purif 16:109–119. doi:
  24. Tyler RC, Sreenath HK, Singh S, Aceti DJ, Bingman CA, Markley JL, Fox BG (2005) Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination. Protein Expr Purif 40:268–278. doi:
  25. Wang W, Sun J, Nimtz M, Deckwer WD, Zeng AP (2003) Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences. Proteome Sci 1:6. doi:
  26. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4:171–180. doi:
  27. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81. doi:
  28. Yamazaki T, Lee W, Arrowsmith CH, Muhandiram DR, Kay LE (1994) A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J Am Chem Soc 116:11655–11666. doi:
  29. Zahn R, von Schroetter C, Wuthrich K (1997) Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding. FEBS Lett 417:400–404. doi:
  30. Zhao Q, Frederick R, Seder K, Thao S, Sreenath H, Peterson F, Volkman BF, Markley JL, Fox BG (2004) Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N. J Struct Funct Genomics 5:87–93. doi:
  31. Zhao KQ, Hartmett J, Slater MR (2007) Selenomethionine protein labeling using the Escherichia coli strain KRX. Promega Notes 96:24–26
• 作者单位：1. Helmholtz Centre for Infection Research (SB), Braunschweig, Germany2. Technical Chemistry - Life Science, Leibniz University of Hannover, Hannover, Germany
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins (15N, 13C, and 2H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15N and 13C, respectively, and 75% of (non-exchangeable) hydrogen for 2H labeling. The expression yields and final cell densities (OD600 ∼16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.