Noncytotoxic DsRed derivatives for whole-cell labeling.
Fluorescent proteins (FPs) are invaluable tools for biomedical research. Useful FPs have desirable fluorescence properties such as brightness and photostability, but a limitation is that many orange, red, and far-red FPs are cytotoxic when expressed in the cytosol. This cytotoxicity stems from aggregation. To reduce aggregation, we engineered the surface of DsRed-Express to generate DsRed-Express2, a highly soluble tetrameric FP that is noncytotoxic in bacterial and mammalian cells. Directed evolution of DsRed-Express2 yielded the color variants E2-Orange, E2-Red/Green, and E2-Crimson. These variants can be used to label whole cells for single- and multi-color experiments employing microscopy or flow cytometry. Methods are described for reducing the higher-order aggregation of oligomeric FPs and for analyzing FP cytotoxicity in Escherichia coli and HeLa cells.
Department of Biochemistry and Molecular Biology, Gordon Center for Integrated Sciences, The University of Chicago, Chicago, IL, USA.
Rita L
RL
Strack
Robert J
RJ
Keenan
Benjamin S
BS
Glick
D002470:Cell Survival; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein