Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride.
The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.
Ishii
Marina
M
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, SP, Brazil. marishii@usp.br
Kunimura
Juliana Sayuri
JS
Jeng
Hélio Tallon
HT
Penna
Thereza Christina Vessoni
TC
Cholewa
Olivia
O
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D015336:Molecular Probe Techniques; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
D011994:Recombinant Proteins; D012996:Solutions; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride