Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

Abstract

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

Quantitative assessment of near-infrared fluorescent proteins

“…Expanding spectral diversity of FPs into nearinfrared (NIR) range (~650-900 nm) of the electromagnetic spectrum enabled new imaging capabilities ranging from multiplexed super-resolution imaging of live cells to whole-body in vivo imaging 2 . Red shifted uorescence spectrum of NIR FPs allows for e cient excitation with standard red lasers (630-640 nm), widely used light sources in microscopy, unlocking standard Cy5 lter set for FPs, which previously were used only for red-shifted dye imaging [3][4][5][6] . Correspondingly, NIR FPs can be readily imaged with other conventional FPs such as GFPs and RFPs enabling multiplexed structural 6 and functional imaging [7][8][9] .…”

“…Red shifted uorescence spectrum of NIR FPs allows for e cient excitation with standard red lasers (630-640 nm), widely used light sources in microscopy, unlocking standard Cy5 lter set for FPs, which previously were used only for red-shifted dye imaging [3][4][5][6] . Correspondingly, NIR FPs can be readily imaged with other conventional FPs such as GFPs and RFPs enabling multiplexed structural 6 and functional imaging [7][8][9] .…”

“…Further development of monomeric NIR FPs enabled their application as uorescent tags for subcellular structures in cultured cells under conventional 6,10 and super-resolution microscopy [11][12][13] . In addition, NIR FPs are particularly bene cial for in vivo imaging of model organisms due to reduced auto uorescence, low light scattering, and minimal absorbance of tissue at longer wavelengths 14,15 .…”

“…However, the majority of currently available NIR FPs are derived from bacteriophytochromes, which utilize a linear tetrapyrrole biliverdin IXα (BV) as a chromophore 14 . This feature makes the uorescence of bacteriophytochrome photoreceptor (BphP)-based NIR FPs dependent on the heme metabolism in host cells 6,8,18 . As a result, the performance of BphP-based NIR FPs in cultured cells and in vivo preparations cannot be predicted based on the in vitro characteristics measured on proteins puri ed from bacteria, which is the most common way to select FPs for an imaging experiment 6,13 .…”

“…This feature makes the uorescence of bacteriophytochrome photoreceptor (BphP)-based NIR FPs dependent on the heme metabolism in host cells 6,8,18 . As a result, the performance of BphP-based NIR FPs in cultured cells and in vivo preparations cannot be predicted based on the in vitro characteristics measured on proteins puri ed from bacteria, which is the most common way to select FPs for an imaging experiment 6,13 . Furthermore, intracellular brightness of BphP-based NIR FPs in one cell type is not necessarily retained in another cell type 6 .…”