Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values.
The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5-9.0 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees C) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85 degrees C, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate). The stability of GFPuv in Tris-HCl (pH >8.0) was constant at 90 and 95 degrees C, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100 degrees C.
Penna
Thereza Christina Vessoni
TC
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Rua Antonio de Macedo Soares, 452, 04607-000, São Paulo/SP, Brazil. tcvpenna@usp.br
Ishii
Marina
M
Junior
Adalberto Pessoa
AP
Cholewa
Olivia
O
D000085:Acetates / Q000737:chemistry; D001709:Biotechnology / Q000379:methods*; D002021:Buffers; D002845:Chromatography; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D000085:Acetates; D002021:Buffers; D008164:Luminescent Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins