Protein Engineering, Vol. 15, No. 9, 763-769,
September 2002
© 2002 Oxford University Press
Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein
1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, 2 Structural Biology Group, Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, 3 Department of Biology, Kyushu University, Fukuoka 812-8581, 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 101-0062 and 5 Department of Structural Biology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The 1H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929–17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, 15N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone 15N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.