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Applied Biochemistry and Biotechnology

, Volume 122, Issue 1–3, pp 501–527 | Cite as

Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values

  • Thereza Christina Vessoni PennaEmail author
  • Marina Ishii
  • Juliana Sayuri Kunimura
  • Olivia Cholewa
Article

Abstract

The stability at room temperature (25°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.

Index Entries

Protein stability decimal reduction time green fluorescent protein glucose solution denaturation conductivity 

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Thereza Christina Vessoni Penna
    • 1
    Email author
  • Marina Ishii
    • 1
  • Juliana Sayuri Kunimura
    • 1
  • Olivia Cholewa
    • 2
  1. 1.Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical ScienceUniversity of São PauloSPBrazil
  2. 2.Molecular Probes, Inc.Eugene

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