Table 3.
Properties of GFP derivatives used for setup of the mutational library and variants with improved in vivo expression in thermophilic bacilli
| GFP | Changes to A. victoria wild-type GFPa | Properties | Optimization method | References |
|---|---|---|---|---|
| sfGFP(Sp) | S30R, Y39N, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A206V | Used for random mutagenesis and FACS library construction in DSM 2542 in this study | S. pneumoniae using OPTIMIZER program | [35] |
| sfGFP(N39D/A179A) | S30R, N39D, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A179A, A206V | Improved in vivo thermostability and brightness (885-fold) in comparison to sfGFP(Sp) at 60 °C | Random mutagenesis and cell sorting by FACS | This study |
| sfGFP(N39D/A179A/H231H) | S30R, N39D, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A179A, A206V, H231H | Improved in vivo thermostability and brightness (310-fold) in comparison to sfGFP(Sp) at 60 °C | Directed mutagenesis by introduction of silent mutation H231H into sfGFP(N39D/A179A) | This study |
| sfGFP(N39D/Y66 W/A179A) | S30R, N39D, F64L, S65T, Y66W, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A179A, A206V | Cyan variant with improved in vivo thermostability in thermophilic bacilli | Directed mutagenesis by introduction of cyan color mutation Y66 W into sfGFP(N39D/A179A) | This study |
| sfGFP(N39D/A179A/T203Y) | S30R, N39D, F64L, S65T, Q80R, F99S, N105T, Y145F, M153T, V163A, I171V, A179A, T203Y, A206V | Yellow variant with improved in vivo thermostability in thermophilic bacilli | Directed mutagenesis by introduction of yellow color mutation T203Y into sfGFP(N39D/A179A) | This study |
aItalicized amino acid exchanges indicate changes to the sfGFP(Sp) protein