Engineering and characterization of new LOV-based fluorescent proteins from chlamydomonas reinhardtii and vaucheria frigida

Arnab Mukherjee, Kevin B. Weyant, Utsav Agrawal, Joshua Walker, Isaac K.O. Cann, Charles M. Schroeder

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)371-377
Number of pages7
JournalACS synthetic biology
Volume4
Issue number4
DOIs
StatePublished - Apr 17 2015

Keywords

  • LOV domains
  • flavin-based fluorescent proteins
  • fluorescent reporters
  • genome mining

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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