Evaluation of the pH- and Thermal Stability of the Recombinant Green Fluorescent Protein (GFP) in the Presence of Sodium Chloride

• Published in: Applied biochemistry and biotechnology 2007-04, Vol.137-140 (1-12), p.555-571
• Main Authors: ISHII, Marina, SAYURI KUNIMURA, Juliana, TALLON JENG, Helio, VESSONI PENNY, Thereza Christina, CHOLEWA, Olivia
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Hydrogen-Ion Concentration; Molecular Probe Techniques; Protein Denaturation; Proteins; Recombinant Proteins - chemistry; Salt; Sodium chloride; Sodium Chloride - chemistry; Solutions; Spectrometry, Fluorescence - methods; Studies; Temperature
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-007-9079-6

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.