Functional immobilization and patterning of proteins by an enzymatic transfer reaction.
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.
Waichman
Sharon
S
Division of Biophysics, University of Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany.
Bhagawati
Maniraj
M
Podoplelova
Yulia
Y
Reichel
Annett
A
Brunk
Ariane
A
Paterok
Dirk
D
Piehler
Jacob
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D016898:Interferon-alpha / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D053633:Receptor, Interferon alpha-beta / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013499:Surface Properties; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*
D001426:Bacterial Proteins; D055767:Immobilized Proteins; D016898:Interferon-alpha; D009842:Oligopeptides; C082662:phosphopantetheinyl transferase; D053633:Receptor, Interferon alpha-beta; D011092:Polyethylene Glycols; D010204:Pantetheine; D017855:Transferases (Other Substituted Phosphate Groups); C003129:4'-phosphopantetheine; D003065:Coenzyme A