Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method

  • Yoshihiro Kobashigawa
  • Hiroyuki Kumeta
  • Kenji Ogura
  • Fuyuhiko Inagaki


Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus.


Sortase Protein ligation GB1 Intein INSET Solubility enhancement 



Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Isotopically invisible solubility/stability enhancement tag



This work was supported by Grants-in-Aid for Scientific Research and the National Project on “Targeted Proteins Research Program” from the Ministry of Education, Science and Culture of Japan.

Supplementary material

10858_2008_9296_MOESM1_ESM.pdf (433 kb)
Supplementary material 1 (PDF 432 kb)


  1. Bagby S, Tong KI, Liu D, Alattia JR, Ikura M (1997) The button test: a small scale method using microdialysis cells for assessing protein solubility at concentrations suitable for NMR. J Biomol NMR 10:279–282CrossRefGoogle Scholar
  2. Camarero JA, Shekhtman A, Campbell EA, Chlenov M, Gruber TM, Bryant DA, Darst SA, Cowburn D, Muir TW (2002) Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 99:8536–8541CrossRefADSGoogle Scholar
  3. Chang YG, Song AX, Gao YG, Shi YH, Lin XJ, Cao XT, Lin DH, Hu HY (2006) Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin. Protein Sci 15:1248–1259CrossRefGoogle Scholar
  4. Gronenborn AM, Filpula DR, Essig NZ, Achari A, Whitlow M, Wingfield PT, Clore GM (1991) A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253:657–661CrossRefADSGoogle Scholar
  5. Huang B, Eberstadt M, Olejniczak ET, Meadows RP, Fesik SW (1996) NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain. Nature 384:638–641CrossRefADSGoogle Scholar
  6. Huth JR, Bewley CA, Jackson BM, Hinnebusch AG, Clore GM, Gronenborn AM (1997) Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. Protein Sci 6:2359–2364CrossRefGoogle Scholar
  7. Kobashigawa Y, Naito M, Inagaki F (2007a) An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates. J Biotechnol 131:458–465CrossRefGoogle Scholar
  8. Kobashigawa Y, Sakai M, Naito M, Yokochi M, Kumeta H, Makino Y, Ogura K, Tanaka S, Inagaki F (2007b) Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK. Nat Struct Mol Biol 14:503–510CrossRefGoogle Scholar
  9. Mao H, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126:2670–2671CrossRefGoogle Scholar
  10. Mazmanian SK, Liu G, Ton-That H, Schneewind O (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285:760–763CrossRefGoogle Scholar
  11. Muralidharan V, Muir TW (2006) Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Method 3:429–438CrossRefGoogle Scholar
  12. Novick RP (2000) Sortase: the surface protein anchoring transpeptidase and the LPXTG motif. Trends Microbiol 8:148–151CrossRefGoogle Scholar
  13. Nyanguile O, Dancik C, Blakemore J, Mulgrew K, Kaleko M, Stevenson SC (2003) Synthesis of adenoviral targeting molecules by intein-mediated protein ligation. Gene Ther 10:1362–1369CrossRefGoogle Scholar
  14. Otomo T, Teruya K, Uegaki K, Yamazaki T, Kyogoku Y (1999) Improved segmental isotope labeling of proteins and application to a larger protein. J Biomol NMR 14:105–114CrossRefGoogle Scholar
  15. Perry AM, Ton-That H, Mazmanian SK, Schneewind O (2002) Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring. PXTG motif. J Biol Chem 277:16241–16248CrossRefGoogle Scholar
  16. Safadi SS, Shaw GS (2007) A disease state mutation unfolds the parkin ubiquitin-like domain. Biochemistry 46:14162–14169CrossRefGoogle Scholar
  17. Sugase K, Landes MA, Wright PE, Martinez-Yamout M (2008) Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes. Protein Expres Purif 57:108–115CrossRefGoogle Scholar
  18. Xu R, Ayers B, Cowburn D, Muir TW (1999) Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies. Proc Natl Acad Sci USA 96:388–393CrossRefADSGoogle Scholar
  19. Yamazaki T, Otomo T, Oda N, Kyogoku Y, Uegaki K, Ito N, Ishino Y, Nakamura H (1998) Segmental isotope labeling for protein NMR using peptide splicing. J Am Chem Soc 120:5591–5592CrossRefGoogle Scholar
  20. Zhou P, Lugovskoy AA, Wagner G (2001) A solubility-enhancement tag (SET) for NMR studies of poor behaving proteins. J Biomol NMR 20:11–14CrossRefGoogle Scholar
  21. Züger S, Iwai H (2005) Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nat Biotechnol 23:736–740CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Yoshihiro Kobashigawa
    • 1
  • Hiroyuki Kumeta
    • 1
  • Kenji Ogura
    • 1
  • Fuyuhiko Inagaki
    • 1
  1. 1.Department of Structural Biology, Graduate School of Pharmaceutical SciencesHokkaido UniversitySapporoJapan

Personalised recommendations