Variants of green fluorescent protein GFPxm.
As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could produce strong fluorescence when expressed at 37 degrees C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37 degrees C, and mutants GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42 degrees C. Meanwhile, at least 4 mutants could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak.
Luo
Wen-Xin
WX
National Institute of Diagnostics and Vaccine Development in infectious disease, The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen, People's Republic of China.
Cheng
Tong
T
Guan
Bao-Quan
BQ
Li
Shao-Wei
SW
Miao
Ji
J
Zhang
Jun
J
Xia
Ning-Shao
NS
D000818:Animals; D045744:Cell Line, Tumor; D032621:DNA Shuffling; D005453:Fluorescence; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*
D050505:Mutant Proteins; D049452:Green Fluorescent Proteins