title: SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase.
abstract: SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC increased the thermodynamic stability of the structure and remained more properly folded at high temperatures when compared with the wild type. We also prepared an N-terminally and C-terminally shortened form of the SpyCatcher protein and cyclization using this truncated form led to even more thermostability than the original form. Our findings suggest that cyclization with SpyTag and SpyCatcher is a promising and effective strategy to enhance thermostability of enzymes.
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Si
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, China.|Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.affiliation: College of Food Sciences and Light Industry, Nanjing Tech University, Nanjing, 211816, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, China.|School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China.
authors.forename: He
authors.identifier: http://orcid.org/0000-0003-4670-9018
authors.initials: H
authors.lastname: Huang