Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase |
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Authors: | Chong Zhang Min-Sheng Liu Xin-Hui Xing |
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Institution: | (1) Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing, 100084, People’s Republic of China |
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Abstract: | By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic
xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase
have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the
thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence
intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered
by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to
xylanase activity (R
gfp/A
xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R
gfp/A
xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the
correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio
of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could
be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins. |
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Keywords: | Temperature Green fluorescent protein Fusion protein Hyperthermophilic xylanase Fluorescence intensity |
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