Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values.
The stability at room temperature (25 degrees C) of recombinant green fluorescent protein (GFPuv), expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction column, was studied. The GFPuv was diluted in buffered (each 10 mM: Tris-HCl, pH 8.0; phosphate, pH 6.0 and 7.0 and acetate, pH 5.0) and in unbuffered (water for injection [WFI]; pH 6.70 +/- 0.40) glucose solutions (from 1.5 to 50%). By assaying the loss of fluorescence intensity as a measure of denaturation, the stability of GFPuv in these solutions was evaluated relative to glucose concentration, pH, osmolarity, density, conductivity, and viscosity. The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time required to reduce 90% of the initial fluorescence intensity of GFPuv. The D-value between 56 and 83 h of GFPuv at 1.5-15% glucose in WFI was equivalent to 20-30% glucose in a phosphate. The stability of GFPuv in 50% glucose was similar for all buffers studied and four times higher than in WFI. By the convenient measure of fluorescence intensity, GFPuv can be used as an indicator to report the extent of denaturation rates of other proteins in glucose solutions.
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, SP, Brazil. tcvpenna@usp.br
Ishii
Marina
M
Kunimura
Juliana Sayuri
JS
Cholewa
Olivia
O
D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000032:analysis / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014783:Viscosity
D011994:Recombinant Proteins; D012996:Solutions; D049452:Green Fluorescent Proteins; D005947:Glucose