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

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°C). From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (lg ml )1 ). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (E a ) were calculated. Conclusions: For temperatures between 45 and 70°C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70°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.

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride

“…In our previous work, the presence of NaCl into solutions improved GFP thermal stability compared with glucose solutions made with the same buffers used in this work (11). The buffered glucose/NaCl solutions at pH 7.0 provided the highest D-values (D 90°C = 6.52 min and D 95°C = 4.93 min), equivalent for glucose/Tris-EDTA buffered solution (pH 8.0; D 90°C = 6.64 min and D 95°C = 5.20 min), for the same concentration of glucose, proving that the addition of glucose and NaCl in the same solution affected GFP thermal stability favorably.…”

“…The expression of recombinant GFP by E. coli DH5-α, the extraction and purification of GFP have been outlined in previous experiments (8)(9)(10)(11)(12). GFP fluorescence intensity was measured in a spectrofluorometer (λ Excitation = 394 nm, λ Emission = 509 nm) (RF 5301 PC; Shimadzu Corporation, Kyoto, Japan).…”

Resistência de microrganismos presentes em ambiente hospitalar e sistema de purificação de água e uso de proteína verde fluorescente (GFP) como potencial indicador biológico

“…A proteína verde florescente recombinante expressada por Escherichia coli foi extraída pelo método de partição em três fases (TPP) e purificada através de coluna butil de interação hidrofóbica (VESSONI PENNA et al, 2002a, VESSONI PENNA et al, 2002b, VESSONI PENNA et al, 2004a. A intensidade de fluorescência da GFP extraída foi determinada em espectrofluorímetro (Shimadzu Co., Kyoto, Japão), em λ EX =394 nm e λ EM =509 nm).…”

“…A proteína verde fluorescente, que apresenta excitação máxima em 395 nm e emissão máxima em 509 nm, é um monômero compacto e globular de 27kDa, de características ácidas (pI 4,6-5,4), resistente ao calor (T≥100 o C) (VESSONI PENNA et al, 2004a, b), pH alcalino (até 12, sendo valor de pH ótimo entre 6 e 8) (VESSONI PENNA et al, 2002a, b), presença de sais, e agentes químicos (PENNA et al, 2004a,b, WARD, 2006. A estabilidade da GFP pode ser relacionada com a fluorescência emitida, baseado na estrutura do fluoróforo (FÁGÁIN, 1995, MOZHAEV E MARTIEK, 1982, PENNA et al, 2004a, a intensidade de fluorescência pode ser quantificada rapidamente in situ utilizando método espectrofluorimétrico.…”

“…A estabilidade da GFP pode ser relacionada com a fluorescência emitida, baseado na estrutura do fluoróforo (FÁGÁIN, 1995, MOZHAEV E MARTIEK, 1982, PENNA et al, 2004a, a intensidade de fluorescência pode ser quantificada rapidamente in situ utilizando método espectrofluorimétrico.…”