Enhanced small green fluorescent proteins as a multisensing platform for biosensor development

Guo-Teng Liang; Cuixin Lai; Zejun Yue; Hanbin Zhang; Danyang Li; Zhong Chen; Xingyu Lu; Liang Tao; Fedor V Subach; Kiryl D Piatkevich

doi:10.3389/fbioe.2022.1039317

Enhanced small green fluorescent proteins as a multisensing platform for biosensor development

Front Bioeng Biotechnol. 2022 Oct 17:10:1039317. doi: 10.3389/fbioe.2022.1039317. eCollection 2022.

Authors

Guo-Teng Liang^{1

2

3

4}, Cuixin Lai^{1

2

3}, Zejun Yue^{1

2

3

5}, Hanbin Zhang^{1

2

3}, Danyang Li^{1

2

3}, Zhong Chen⁶, Xingyu Lu⁶, Liang Tao^{1

2

3}, Fedor V Subach⁷, Kiryl D Piatkevich^{1

2

3}

Affiliations

¹ School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.
² Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China.
³ Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China.
⁴ Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China.
⁵ School of Basic Medical Sciences, Xi'an Jiao Tong University, Xi'an, Shaanxi, China.
⁶ Key Laboratory of Precise Synthesis of Functional Molecules of Zhejiang Province, School of Science, Instrumentation and Service Center for Molecular Sciences, Westlake University, Hangzhou, Zhejiang, China.
⁷ Complex of NBICS Technologies, National Research Center "Kurchatov Institute", Moscow, Russia.

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 Escherichia coli and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs' fluorescence was highly sensitive to Cu(II) ions in solution with K_d values of 67 and 68 nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for in vitro and in-cell applications.

Keywords: copper; flavin; fluorescent protein biosensor; hypoxia; live cell imaging microscopy.