Kamiya Noriho, Abe Hiroki, Goto Masahiro, Tsuji Yukiko, Jikuya Hiroyuki
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
Org Biomol Chem. 2009 Sep 7;7(17):3407-12. doi: 10.1039/b904046c. Epub 2009 Jun 30.
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.
设计了具有多种荧光团的新型小底物，用于微生物转谷氨酰胺酶（MTG）催化的蛋白质共价标记。新设计基于MTG在底物识别方面的灵活性，可用于取代传统转谷氨酰胺酶底物苄氧羰基-L-谷氨酰胺基甘氨酸（Z-QG）的N端保护基。在此，我们首次报道，当通过β-丙氨酸或ε-氨基己酸连接子连接时，MTG可以接受多种荧光团（丹磺酰基、荧光素和罗丹明衍生物）来取代苄氧羰基部分。通过对与重组细菌碱性磷酸酶N端融合的含MTG反应性赖氨酸的肽标签进行位点特异性、共价和定量标记，并保留靶蛋白功能，证明了新型荧光底物的实用性。
