title: Sortase A-catalyzed site-specific coimmobilization on microparticles via streptavidin.
abstract: A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG)was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D001710:Biotin; D002384:Catalysis; D003546:Cysteine Endopeptidases; D004800:Enzymes, Immobilized; D008722:Methods; D019809:Streptavidin
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D001710:Biotin; D019809:Streptavidin; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases