Functional immobilization and patterning of proteins by an enzymatic transfer reaction

Anal Chem. 2010 Feb 15;82(4):1478-85. doi: 10.1021/ac902608a.


Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Coenzyme A / metabolism
  • Glass / chemistry
  • Immobilized Proteins / chemistry*
  • Immobilized Proteins / metabolism*
  • Interferon-alpha / chemistry
  • Interferon-alpha / metabolism
  • Models, Molecular
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Pantetheine / analogs & derivatives
  • Pantetheine / metabolism
  • Polyethylene Glycols / chemistry
  • Protein Structure, Tertiary
  • Receptor, Interferon alpha-beta / chemistry
  • Receptor, Interferon alpha-beta / metabolism
  • Spectrum Analysis
  • Substrate Specificity
  • Surface Properties
  • Transferases (Other Substituted Phosphate Groups) / metabolism*


  • Bacterial Proteins
  • Immobilized Proteins
  • Interferon-alpha
  • Oligopeptides
  • phosphopantetheinyl transferase
  • Receptor, Interferon alpha-beta
  • Polyethylene Glycols
  • Pantetheine
  • Transferases (Other Substituted Phosphate Groups)
  • 4'-phosphopantetheine
  • Coenzyme A