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Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability

  • Tingting Lin (School of Life Science, Anhui Agricultural University) ;
  • Yuanyuan Ge (School of Life Science, Anhui Agricultural University) ;
  • Qing Gao (School of Life Science, Anhui Agricultural University) ;
  • Di Zhang (School of Life Science, Anhui Agricultural University) ;
  • Xiaofeng Chen (School of Life Science, Anhui Agricultural University) ;
  • Yafang Hu (School of Life Science, Anhui Agricultural University) ;
  • Jun Fan (School of Life Science, Anhui Agricultural University)
  • Received : 2023.05.11
  • Accepted : 2023.08.17
  • Published : 2023.12.28

Abstract

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

Keywords

Acknowledgement

This study was financially supported by the Anhui Educational Committee Foundation (KJ2020A0113), P.R. China.

References

  1. Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, et al. 2017. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42: 111-129. https://doi.org/10.1016/j.tibs.2016.09.010
  2. Mukherjee A, Schroeder CM. 2015. Flavin-based fluorescent proteins: emerging paradigms in biological imaging. Curr. Opin. Biotechnol. 31: 16-23. https://doi.org/10.1016/j.copbio.2014.07.010
  3. Buckley AM, Petersen J, Roe AJ, Douce GR, Christie JM. 2015. LOV-based reporters for fluorescence imaging. Curr. Opin. Chem. Biol. 27: 39-45. https://doi.org/10.1016/j.cbpa.2015.05.011
  4. Wingen M, Jaeger KE, Gensch T, Drepper T. 2017. Novel thermostable flavin-binding fluorescent proteins from thermophilic organisms. Photochem. Photobiol. 93: 849-856. https://doi.org/10.1111/php.12740
  5. Mukherjee A, Weyant KB, Agrawal U, Walker J, Cann IK, Schroeder CM. 2015. Engineering and characterization of new LOV-based fluorescent proteins from Chlamydomonas reinhardtii and Vaucheria frigida. ACS Synth. Biol. 4: 371-377. https://doi.org/10.1021/sb500237x
  6. Close DW, Paul CD, Langan PS, Wilce MC, Traore DA, Halfmann R, et al. 2015. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering. Proteins 83: 1225-1237. https://doi.org/10.1002/prot.24699
  7. Cardoso Ramos F, Cupellini L, Mennucci B. 2021. Computational investigation of structural and spectroscopic properties of LOV-based proteins with improved fluorescence. J. Phys. Chem. B 125: 1768-1777. https://doi.org/10.1021/acs.jpcb.0c10834
  8. Mishra A, Sharma A, Kateriya S. 2023. Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: a combined molecular dynamics simulation and biophysical approach. Biochim. Biophys. Acta Gen. Subj. 1867: 130304.
  9. Ko S, Hwang B, Na JH, Lee J, Jung ST. 2019. Engineered Arabidopsis blue light receptor LOV domain variants with improved quantum yield, brightness, and thermostability. J. Agric. Food Chem. 67: 12037-12043. https://doi.org/10.1021/acs.jafc.9b05473
  10. Liang GT, Lai C, Yue Z, Zhang H, Li D, Chen Z, et al. 2022. Enhanced small green fluorescent proteins as a multisensing platform for biosensor development. Front. Bioeng. Biotechnol. 10: 1039317.
  11. Mukherjee A, Weyant KB, Walker J, Schroeder CM. 2012. Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida. J. Biol. Eng. 6: 20.
  12. He X, Zhang S, Dang D, Lin T, Ge Y, Chen X, et al. 2023. Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle. Microb. Cell Fact. 22: 2.
  13. Baird GS, Zacharias DA, Tsien RY. 2000. Biochemistry, mutagenesis, and oligomerization of DSRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97: 11984-11989. https://doi.org/10.1073/pnas.97.22.11984
  14. Sarmiento C, Camarero JA. 2019. Biotechnological applications of protein splicing. Curr. Protein Pept. Sci. 20: 408-424. https://doi.org/10.2174/1389203720666190208110416
  15. Scott CP, Abel-Santos E, Wall M, Wahnon DC, Benkovic SJ. 1999. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. USA 96: 13638-13643. https://doi.org/10.1073/pnas.96.24.13638
  16. Zhao Z, Ma X, Li L, Zhang W, Ping S, Xu MQ, et al. 2010. Protein cyclization enhanced thermostability and exopeptidase-resistance of green fluorescent protein. J. Microbiol. Biotechnol. 20: 460-466.
  17. Ciragan A, Aranko AS, Tascon I, Iwai H. 2016. Salt-inducible protein splicing in cis and trans by inteins from extremely Halophilic archaea as a novel protein-engineering tool. J. Mol. Biol. 428: 4573-4588. https://doi.org/10.1016/j.jmb.2016.10.006
  18. Stevens AJ, Sekar G, Shah NH, Mostafavi AZ, Cowburn D, Muir TW. 2017. A promiscuous split intein with expanded protein engineering applications. Proc. Natl. Acad. Sci. USA 114: 8538-8543. https://doi.org/10.1073/pnas.1701083114
  19. Callahan BP, Stanger M, Belfort M. 2013. A redox trap to augment the intein toolbox. Biotechnol. Bioeng. 110: 1565-1573. https://doi.org/10.1002/bit.24821
  20. Lin T, Zhang S, Zhang D, Chen X, Ge Y, Hu Y, et al. 2023. Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein. Protein Expr. Purif. 14: 106272.
  21. Zhou C, Yan Y, Fang J, Cheng B, Fan J. 2014. A new fusion protein platform for quantitatively measuring activity of multiple proteases. Microb. Cell Fact. 13: 44.
  22. Xiao W, Jiang L, Wang W, Wang R, Fan J. 2018. Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli. J. Biosci. Bioeng. 125: 160-167. https://doi.org/10.1016/j.jbiosc.2017.08.016
  23. Kurz M, Cowieson NP, Robin G, Hume DA, Martin JL, Kobe B, et al. 2006. Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins. Protein Expr. Purif. 50: 68-73. https://doi.org/10.1016/j.pep.2006.05.006
  24. Zou W, Nguyen HN, Zastrow ML. 2022. Mutant flavin-based fluorescent protein sensors for detecting intracellular zinc and copper in Escherichia coli. ACS Sens. 7: 3369-3378. https://doi.org/10.1021/acssensors.2c01376
  25. Nikolaev A, Yudenko A, Smolentseva A, Bogorodskiy A, Tsybrov F, Borshchevskiy V, et al. 2023. Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging. J. Biol. Chem. 299: 102977.
  26. Losi A, Gartner W, Raffelberg S, Cella Zanacchi F, Bianchini P, Diaspro A, et al. 2013. A photochromic bacterial photoreceptor with potential for super-resolution microscopy. Photochem. Photobiol. Sci. 12: 231-235. https://doi.org/10.1039/c2pp25254f
  27. Yudenko A, Smolentseva A, Maslov I, Semenov O, Goncharov IM, Nazarenko VV, et al. 2021. Rational design of a split flavin-based fluorescent reporter. ACS Synth. Biol. 10: 72-83. https://doi.org/10.1021/acssynbio.0c00454
  28. Homans RJ, Khan RU, Andrews MB, Kjeldsen AE, Natrajan LS, Marsden S, et al. 2018. Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV. Phys. Chem. Chem. Phys. 20: 16949-16955. https://doi.org/10.1039/C8CP01699B
  29. Iwai H, Pluckthun A. 1999. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459: 166-172. https://doi.org/10.1016/S0014-5793(99)01220-X
  30. Nandy S, Maranholkar VM, Crum M, Wasden K, Patil U, Goyal A, et al. 2023. Expression and characterization of intein-cyclized trimer of Staphylococcus aureus protein A domain Z. Int. J. Mol. Sci. 24: 1281.
  31. Iwai H, Lingel A, Pluckthun A. 2001. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276: 16548-16554. https://doi.org/10.1074/jbc.M011639200
  32. Boassa D, Lemieux SP, Lev-Ram V, Hu J, Xiong Q, Phan S, et al. 2019. Split-miniSOG for spatially detecting intracellular protein-protein interactions by correlated light and electron microscopy. Cell Chem. Biol. 26: 1407-1416. https://doi.org/10.1016/j.chembiol.2019.07.007
  33. Nazarenko VV, Remeeva A, Yudenko A, Kovalev K, Dubenko A, Goncharov IM, et al. 2019. A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies. Photochem. Photobiol. Sci. 18: 1793-1805. https://doi.org/10.1039/c9pp00067d
  34. Halavaty AS, Moffat K. 2007. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry 46: 14001-14009. https://doi.org/10.1021/bi701543e
  35. Ojima-Kato T, Nagai S, Nakano H. 2017. N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae. J. Biosci. Bioeng. 123: 540-546. https://doi.org/10.1016/j.jbiosc.2016.12.004
  36. Cava F, de Pedro MA, Blas-Galindo E, Waldo GS, Westblade LF, Berenguer J. 2008. Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology. Environ. Microbiol. 10: 605-613. https://doi.org/10.1111/j.1462-2920.2007.01482.x
  37. Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW. 2016. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138: 2162-2165. https://doi.org/10.1021/jacs.5b13528
  38. Xu Y, Zhang L, Ma B, Hu L, Lu H, Dou T, et al. 2018. Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE. Enzyme Microb. Technol. 118: 6-12. https://doi.org/10.1016/j.enzmictec.2018.06.013