title: Engineering and characterization of new LOV-based fluorescent proteins from Chlamydomonas reinhardtii and Vaucheria frigida.
abstract: Flavin-based fluorescent proteins (FbFPs) are a new class of fluorescent reporters that exhibit oxygen-independent fluorescence, which is a key advantage over the green fluorescent protein. Broad application of FbFPs, however, has been generally hindered by low brightness. To maximize the utility of FbFPs, there is a pressing need to expand and diversify the limited FbFP library through the inclusion of bright and robust variants. In this work, we use genome mining to identify and engineer two new FbFPs (CreiLOV and VafLOV) from Chlamydomonas reinhardtii and Vaucheria frigida. We show that CreiLOV is a thermostable, photostable, and fast-maturing monomeric reporter that outperforms existing FbFPs in brightness and operational pH range. Furthermore, we show that CreiLOV can be used to monitor dynamic gene expression in Escherichia coli. Overall, our work introduces CreiLOV as a robust addition to the FbFP repertoire and highlights genome mining as a powerful approach to engineer improved FbFPs.
authors.forename: Arnab
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Weyant
authors.forename: Utsav
authors.initials: U
authors.lastname: Agrawal
authors.forename: Joshua
authors.initials: J
authors.lastname: Walker
authors.forename: Isaac K O
authors.initials: IK
authors.lastname: Cann
authors.forename: Charles M
authors.initials: CM
authors.lastname: Schroeder
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D058009:Stramenopiles / Q000235:genetics*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: LOV domains; flavin-based fluorescent proteins; fluorescent reporters; genome mining