[Orange fluorescent protein--modification of green fluorescent protein GFPxm].
Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield.
Luo
Wen-Xin
WX
Ministry of Education, Cell Biology and Tumor Cell Engineering Laboratory, Xiamen University, Xiamen 361005, China.
Chen
Min
M
Cheng
Tong
T
Guan
Bao-Quan
BQ
Li
Shao-Wei
SW
Li
Shao-Jing
SJ
Zhang
Jun
J
Xia
Ning-Shao
NS
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D013696:Temperature
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins