Split Spy0128 as a potent scaffold for protein cross-linking and immobilization

Bioconjug Chem. 2013 Feb 20;24(2):242-50. doi: 10.1021/bc300606b. Epub 2013 Feb 6.


Site-specific cross-linking techniques between proteins and additional functional groups have become increasingly important for expanding the utility of proteins in biochemistry and biotechnology. In order to explore powerful techniques for practical bioconjugation applications, we have validated a technique mediated by a unique property of Streptcoccus pyogenes pilin subunit Spy0128, an autocatalytic intramolecular isopeptide formation in Spy0128. Recently, it has been revealed that Spy0128 can be split into two fragments (split-Spy0128 (residues 18-299 of Spy0128) and isopeptag (residues 293-308 of Spy0128)) that were capable of forming an intermolecular covalent complex. We focused on this unique reconstitution property and first studied the bioconjugation of blue and green fluorescent proteins, enabling the direct monitoring of cross-linking reactions by Förster resonance energy transfer (FRET). A fluorescence lifetime study shows that spatial control of two proteins on the Spy0128 scaffold is possible when one protein is fused to the N-terminus of split-Spy0128 and another one is tethered at the N- or C-terminus of the isopeptag. Furthermore, we demonstrated site-specific protein immobilization mediated by the reconstitution of split-Spy0128 and isopeptag. In this case, a split-Spy0128 mutant with a free N-terminal Cys residue was first immobilized onto beads chemically modified with a maleimide group through a Michael addition process. Then, an isopeptagged protein was successfully immobilized onto the split-Spy0128-immobilized beads. These results suggest that Spy0128 is a potent proteinaceous scaffold available for bioconjugation both in solution and at a solid surface.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Escherichia coli / genetics
  • Fimbriae Proteins / chemistry*
  • Fimbriae Proteins / genetics
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Immobilized Proteins / chemistry*
  • Immobilized Proteins / genetics
  • Molecular Sequence Data
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Streptococcus pyogenes / chemistry*
  • Streptococcus pyogenes / genetics


  • Bacterial Proteins
  • Immobilized Proteins
  • Protein Subunits
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Fimbriae Proteins