抄録
Microbial transglutaminase (MTG) is a unique enzyme that catalyzes the acyl transfer reaction between a primary amine and γ-carboxyamide group of glutamine (Gln) residues in peptides and proteins. When the ε-amino group of lysine (Lys) residues in proteins act as an acyl-acceptor, ε-(γ-Gln)Lys bond is formed. In this study, we have investigated the MTG-mediated site-specific immobilization of an enzyme tagged with a specific peptide linker. We used Escherichia coli alkaline phosphatase (AP) as a model enzyme and β-casein as a template for immobilization because β-casein is readily cross-linked by MTG, suggesting that it has several MTG recognition sites (i.e., reactive Gln and Lys residues). As wild-type AP was not recognized by MTG, two designed specific peptides of which amino acid sequences were MKHKGS and MKHKGGGSGGGSGS, abbreviated as K6-tag and K14-tag respectively, were genetically attached to the N-terminus of AP for MTG-mediated conjugation. We have succeeded in the MTG-mediated site-specific immobilization of the resultant K6- and K14-tagged APs into β-casein matrices chemically created on an agarose support. Results obtained here indicate a potential utility of MTG in functional immobilization of proteins.
