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

Functional immobilization and patterning of proteins by an enzymatic transfer reaction.

Author information

1
Division of Biophysics, University of Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany.

Abstract

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.

PMID:
20092261
DOI:
10.1021/ac902608a
[Indexed for MEDLINE]

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