title: Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values.
abstract: 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.
authors.affiliation: 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
authors.forename: Thereza Christina Vessoni
authors.initials: TC
authors.lastname: Penna
authors.forename: Marina
authors.initials: M
authors.lastname: Ishii
authors.forename: Adalberto Pessoa
authors.initials: AP
authors.lastname: Junior
authors.forename: Olivia
authors.initials: O
authors.lastname: Cholewa
mesh_terms: 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
chemical_list: D000085:Acetates; D002021:Buffers; D008164:Luminescent Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins