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Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida

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13 Pages
English

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Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida . However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes. Results In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence. Conclusions To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.

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Published 01 January 2012
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Mukherjee et al. Journal of Biological Engineering 2012, 6 :20 http://www.jbioleng.org/content/6/1/20
R E S E A R C H Open Access Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida Arnab Mukherjee 1 , Kevin B Weyant 1 , Joshua Walker 1 and Charles M Schroeder 1,2,3*
Abstract Background: Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida . However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes. Results: In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence. Conclusions: To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments. Keywords: Flavin-binding fluorescent proteins, Directed evolution, Site saturation mutagenesis
Background [14]. However, the available palette of GFP-based fluor-Green fluorescent protein (GFP) and related analogs escent proteins is limited by a dependence on molecular have been extensively engineered by directed evolution oxygen, which mediates oxidation of a cyclic tripeptide [1-4] to evolve fluorescent reporters with faster matur- chromophore that is strictly required for fluorescence ation times [5,6], enhanced brightness [7-9], improved [15,16]. In this way, GFP-family reporters require oxygen photostability [10], a wide range of emission wavelengths for fluorescence and do not fluoresce in anaerobic [11,12], enhanced thermal tolerance [13], and improved environments [17-20]. efficiency of Förster resonance energy transfer (FRET) Low oxygen environments are frequently encountered in a broad range of biomedical and industrial biopro-re ondence: cms@ cesses, including bioremediation and fermentation plat-1 *DCeoprartspmentofChemicaillliannodis.BeiodumolecularEngineering,UniversityofIllinois forms for the production of high value reduced a 2 tCeUnrtbearnfao-rCBhiaomphpyaisigcns,aUnrdbaCnoa,mILpu6t1at8i0o1n,alUSBiAology,UniversityofIllinoisat biomolecules ( e.g. , biofuels), hypoxic tissue environments Urbana-Champaign, Urbana, IL 61801, USA that promote tumorigenesis, microbial pathogenesis and Full list of author information is available at the end of the article biofilm development, and in biotechnology applications © 2012 Mukherjee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.