AUTHOR=Liang Guo-Teng , Lai Cuixin , Yue Zejun , Zhang Hanbin , Li Danyang , Chen Zhong , Lu Xingyu , Tao Liang , Subach Fedor V. , Piatkevich Kiryl D. TITLE=Enhanced small green fluorescent proteins as a multisensing platform for biosensor development JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=10 YEAR=2022 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.1039317 DOI=10.3389/fbioe.2022.1039317 ISSN=2296-4185 ABSTRACT=
Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499 nm and a molecular size of ∼13 kDa. We carried out systematic benchmarking of miniGFPs in