Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli

摘要

Aims: The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity. Methods and Results: 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 deg C). From a standard curverelating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (mug ml~(-1)). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), tau-value and energy of activation (E_a) were calculated. Conclusions: For temperatures between 45 and 70 deg C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 deg C, the average decrease in GFPuv fluorescence was between 72 to 83%. Significance and Impact of the Study: 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