Vessoni Penna T C, Ishii M, Cholewa O, de Souza L C
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Brazil.
Lett Appl Microbiol. 2004;38(2):135-9. doi: 10.1111/j.1472-765x.2003.01460.x.
The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity.
GFPuv was expressed by Escherichia coli, extracted by the three-phase partitioning method and purified by elution through an hydrophobic interaction column. The collected fractions were further diluted in Tris-HCl-EDTA (pH 8.0) and subjected to continuous heating at set temperatures (45-95 degrees C). From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (microg ml-1). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (Ea) were calculated.
For temperatures between 45 and 70 degrees C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 degrees C, the average decrease in GFPuv fluorescence was between 72 to 83%.
The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam.
通过分析荧光强度的损失来评估分离提取的重组绿色荧光蛋白（GFPuv）的热稳定性。
GFPuv由大肠杆菌表达，通过三相分配法提取，并通过疏水相互作用柱洗脱纯化。收集的组分进一步用Tris-HCl-EDTA（pH 8.0）稀释，并在设定温度（45 - 95℃）下持续加热。根据荧光强度与GFPuv浓度的标准曲线，将荧光强度的损失转化为变性GFPuv浓度（μg/ml）。为确定GFPuv的热稳定性程度，计算了十进制减少时间（D值）、z值和活化能（Ea）。
对于45至70℃之间的温度，提取的天然GFPuv活性相对于初始天然蛋白浓度下降了11%至75%；高于70℃时，GFPuv荧光的平均下降幅度在72%至83%之间。
GFPuv的热稳定性为其作为荧光生物指示剂评估暴露于蒸汽的液体和材料的处理效果的潜在用途提供了基础。
