Bright Biliprotein Labeling Reaching out to the NIR

Far-red and near infrared emitting chromophores extend the application of fluorescent proteins into the region of maximal transmission of most tissues. We report a new highly bright far-red fluorescent phycobiliprotein, referred to as BDFP1.6, that binds the more readily accessible biliverdin covalently and fluoresces in the far-red (~670 nm). BDFP1.6 has 18.7- and 1.8-fold higher effective brightness and photostability than the reported far-red fluorescent phycobiliprotein, smURFP. While BDFP1.6 has comparable effective brightness to the brightest far-red fluorescent proteins, iRFP670, it has 2.2-fold higher photostability than iRFP670. Furthermore, BDFP1.6 is thermostable up to 80 °C and tolerate acidic conditions down to pH 2. Tandeming BDFP1.6 generated monomeric BDFP1.6:1.6 that covalently binds two biliverdins and has 2.5-folder higher effective brightness than the brightest monomeric far-red fluorescent protein, miRFP670. Far-red BDFP1.6 and near infrared BDFP1.1 1 could biolabel cells in dual colors. Via fusing near infrared BDFP1.1 with far-red 1.2 or 1.6, we constructed BDFP1.1:1.2 or 1.1:1.6, that have a large Stokes shift of 65 nm. Functioning of these BDFPs as biomarkers has been exemplified in E. coli, mammalian cells, and the nematode, Caenorhabditis elegans. REFERENCES 1. Ding, W.-L.; Miao, D.; Hou, Y.-N.; Jiang, S.-P.; Zhao, B.-Q.; Zhou, M.; Scheer, H.; Zhao, K.-H., Small monomeric and highly stable near-infrared fluorescent markers derived of the thermophilic phycobiliprotein, ApcF2. BBA Molecular Cell Research 2017, 1864 (10), 1877-1886.