Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

Hiroshi Kobayashi, G. V.T. Swapna, Kuen Phon Wu, Yuliya Afinogenova, Kenith Conover, Binchen Mao, Gaetano T. Montelione, Masayori Inouye

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS2) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS2-tag is replaced with non-isotope labeled PrS2-tag, silencing the NMR signals from PrS2-tag in isotope-filtered 1H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS 2-tag, fulllength well-behaved RbfA samples could be successfully prepared for NMR studies. PrS2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS2-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS2-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1H, 15N and 13C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1H-15N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

Original languageEnglish (US)
Pages (from-to)303-313
Number of pages11
JournalJournal of biomolecular NMR
Issue number4
StatePublished - Apr 2012

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy


  • Intein
  • Protein NMR
  • Protein S
  • Protein ligation
  • RbfA


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