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.

Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy

“…The established methodology was then utilized to coimmobilize glycine‐tagged glucose oxidase (GOx) and biotinylated horseradish peroxidase (HRP). The functionalized polystyrene particle was capable of detecting glucose since GOx catalyzes the oxidation of glucose to hydrogen peroxide and HRP can oxidize 3,3′,5,5′‐tetramethylbenzidine (TMB) in the presence of H 2 O 2 thereby creating a significant deep blue color 91…”

“…The functionalized polystyrene particle was capable of detecting glucose since GOx catalyzes the oxidation of glucose to hydrogen peroxide and HRP can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H 2 O 2 thereby creating a significant deep blue color. [91] SrtA-mediated transpeptidation has also been used to ligate proteins to other kinds of surfaces apart from beads. LPETGG- Figure 6.…”

“…[109] Regarding the specificity of the glycine nucleophile, it has to be taken into consideration that nonnatural alkylamine derivatives and lysine residues are also potent acyl-acceptor substrates. [77,91] Harrenga et al discovered by SDS-PAGE that the light chain of an L19 Fab fragment containing the sortase recognition motif was covalently cross-linked to the heavy chain in the absence of a glycine nucleophile. The corresponding product formation was suppressed in the presence of lysine or glycine nucleophiles but not in the presence of arginine, and was accelerated at increased pH.…”

A Chemoenzymatic Approach to Protein Immobilization onto Crystalline Cellulose Nanoscaffolds

“…Over the last decade, the transpeptidase sortase A found in Gram‐positive bacteria (where it is used to covalently attach proteins to peptidoglycan layers) has emerged as a powerful tool for the modification of various nature‐derived and engineered molecular constructs 11b,c. 12 This enzyme requires two short recognition sequences and generates a native amide bond upon ligation.…”

Eine chemoenzymatische Kupplungsstrategie zur Immobilisierung von Proteinen auf kristalliner Nanocellulose

“…In den letzten zehn Jahren entwickelte sich die Transpeptidase Sortase A aus Gram‐positiven Bakterien (in denen sie Proteine kovalent mit Peptidoglycan‐Schichten verknüpft) zu einem effektiven Werkzeug für die Konjugation molekularer Konstrukte 11b,c. 12 Dieses Enzym benötigt zwei kurze peptidische Erkennungssequenzen und generiert durch Ligation eine native Amidbindung.…”