Fluorescent protein engineering by in vivo site-directed mutagenesis.
In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided.
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA.
Melvys
M
Valledor
Qinghua
Q
Hu
Paul
P
Schiller
Richard S
RS
Myers
D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli; D005434:Flow Cytometry; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008297:Male; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010957:Plasmids / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D013050:Spectrometry, Fluorescence; D055815:Young Adult
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins