Fluorescent substrates for covalent protein labeling catalyzed by microbial transglutaminase.
Novel small substrates with a variety of fluorophores were designed for the covalent labeling of proteins catalyzed by microbial transglutaminase (MTG). The new design is based on the flexibility in the substrate recognition of MTG for the substitution of the N-terminal protecting group of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG). Here we report for the first time that MTG can accept diverse fluorophores (dansyl, fluorescein, and rhodamine derivatives) in place of the benzyloxycarbonyl moiety when linked via a beta-alanine or epsilon-aminocaproic acid linker. The utility of the new fluorescent substrates was demonstrated by site-specific, covalent and quantitative labeling of an MTG-reactive Lys-containing peptide tag fused to the N-terminus of a recombinant bacterial alkaline phosphatase with retention of target protein functionality.
Kamiya
Noriho
N
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan. nori_kamiya@mail.cstm.kyushu-u.ac.jp
Abe
Hiroki
H
Goto
Masahiro
M
Tsuji
Yukiko
Y
Jikuya
Hiroyuki
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D001426:Bacterial Proteins; D002384:Catalysis; D003432:Cross-Linking Reagents; D004151:Dipeptides; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D011506:Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004151:Dipeptides; D005456:Fluorescent Dyes; D011506:Proteins; C432806:glutaminyl-glycine; D011503:Transglutaminases; D000469:Alkaline Phosphatase