Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli |
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Authors: | Vessoni Penna T C Ishii M Cholewa O de Souza L C |
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Affiliation: | Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of S?o Paulo, Brazil. tcvpenna@usp.br |
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Abstract: | 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 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. CONCLUSIONS: 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%. 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. |
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Keywords: | biological indicator decimal reduction time (D-value) thermal stability recombinant green fluorescent protein (GFPuv) |
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