PUBLICATION

Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

Authors

Babakhanova, S., Jung, E.E., Namikawa, K., Zhang, H., Wang, Y., Subach, O.M., Korzhenevskiy, D.A., Rakitina, T.V., Xiao, X., Wang, W., Shi, J., Drobizhev, M., Park, D., Eisenhard, L., Tang, H., Köster, R.W., Subach, F.V., Boyden, E.S., Piatkevich, K.D.

ID

ZDB-PUB-211217-12

Date

2021

Source

Protein science : a publication of the Protein Society 31(3): 728-751 (Journal)

Registered Authors

Boyden, Edward, Köster, Reinhard W., Namikawa, Kazuhiko

Keywords

none

MeSH Terms

Animals
Green Fluorescent Proteins/genetics
Green Fluorescent Proteins/metabolism
Mice
Mammals/genetics
Fluorescent Dyes/metabolism
Directed Molecular Evolution*
Optical Imaging
Neurons/metabolism
Cell Line
Luminescent Proteins/genetics
Luminescent Proteins/metabolism
Zebrafish*/genetics
Zebrafish*/metabolism

PubMed

34913537 Full text @ Protein Sci.

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 2×10⁷ independent random genes of fluorescent proteins expressed in HEK cells completing one iteration of 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. This article is protected by copyright. All rights reserved.

Genes / Markers

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