Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase.
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 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees 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.
Zhang
Chong
C
Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing, 100084, People's Republic of China.
Liu
Min-Sheng
MS
Xing
Xin-Hui
XH
D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014995:Xylosidases