Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli.
OBJECTIVE
The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity.


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%.


CONCLUSIONS
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.
Vessoni Penna
T C
TC
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Brazil. tcvpenna@usp.br
Ishii
M
M
Cholewa
O
O
de Souza
L C
LC
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006358:Hot Temperature; D007202:Indicators and Reagents; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors; D014169:Transformation, Bacterial
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins