期刊
ANALYTICAL BIOCHEMISTRY
卷 389, 期 2, 页码 138-142出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.03.031
关键词
c-di-GMP; Diguanylate cyclase; Thermophilic; Thermotoga maritima
资金
- Ministry of Education of Singapore
- ARC grant [RG60/06]
- URC grant [RG-151/06]
The cyclic dinucleotide c-di-GMP is a widespread bacterial messenger molecule with potential application as a therapeutic agent for treating bacterial infection. Current enzymatic synthesis of c-di-GMP using mesophilic diguanylate cyclase (DGC) proteins suffers from low production yield due to protein instability and strong product inhibition. Here we report the overexpression and characterization of a stand-alone thermophilic diguanylate cyclase domain (tDGC) protein with enhanced thermostability. The product inhibition that severely limited production yield was significantly alleviated by mutation of a conserved residue in the putative regulatory 1-site. With the mutant tDGC, we demonstrated that hundreds of milligrams of c-di-GMP can be readily prepared by using the optimized procedures for enzymatic reaction and product purification. The thermophilic enzyme will be a valuable tool for other research laboratories for c-di-GMP synthesis as well as the preparation of c-di-GMP derivatives. (C) 2009 Elsevier Inc. All rights reserved.
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