Functional Immobilization and Patterning of Proteins by an Enzymatic Transfer Reaction

Autor(en): Waichman, Sharon
Bhagawati, Maniraj
Podoplelova, Yulia
Reichel, Annett
Brunk, Ariane
Paterok, Dirk
Piehler, Jacob 
Stichwörter: BINDING INTERFACE; Chemistry; Chemistry, Analytical; FUSION PROTEINS; HISTIDINE-TAGGED PROTEINS; I INTERFERON-RECEPTOR; IFNAR1; MOTOR PROTEINS; SFP PHOSPHOPANTETHEINYL TRANSFERASE; SMALL MOLECULES; SURFACES; TECHNOLOGIES
Erscheinungsdatum: 2010
Herausgeber: AMER CHEMICAL SOC
Journal: ANALYTICAL CHEMISTRY
Volumen: 82
Ausgabe: 4
Startseite: 1478
Seitenende: 1485
Zusammenfassung: 
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.
ISSN: 00032700
DOI: 10.1021/ac902608a

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