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.
Similar content being viewed by others
References
Middaugh, C. R. and Volkin, D. B. (1992), in Stability of Protein Pharmaceuticals. Part A. Chemical and Chemical Pathways of Protein Degradation, vol. 2, Ahern, T. J. and Manning, M. C., eds., Plenum, New York, pp. 109–111.
Vojdani, F. (1996), in Methods of Testing Protein Functionality, Hall, G. M., ed., Blackie Academic & Professional, London, pp. 11–59.
Kaushik, J. K. and Bhat, R. (1999), Protein Sci. 8, 222–233.
Sato, S., Sayid, C. J., and Raleigh, D. P. (2000), Protein Sci. 9, 1601–1603.
Timasheff, S. N. (1992), in Stability of Protein Pharmaceuticals. Part B. In Vivo Pathways of Degradation and Strategies for Protein Stabilization, vol. 3, Ahern, T. J. and Manning, M. C., eds., Plenum, New York, pp. 265–285.
Arakawa, T. and Timasheff, S. N. (1982), Biochemistry 21, 6536–6544.
Lee, J. C. and Timasheff, S. N. (1981), J. Biol. Chem. 256(14), 7193–7201.
Kim, Y. S., Jonees, L. S., Dong, A., Kendrick, B. S., Chang, B. S., Manning, M. C., Randolph, T. W., and Carpenter, J. F. (2003), Protein Sci. 12, 1252–1261.
Fágáin, C. Ó. (1995), Biochim. Biophys. Acta 1252, 1–14.
Kulmyrzaev, A., Bryant, C., and McClements, D. J. (2000), J. Agric. Food Chem. 48, 1593–1597.
Semenova, M. G., Antipova, A. S., and Belyakova, L. E. (2002), Curr. Opin. Colloid Interface Sci. 7, 438–444.
Turco, S. J. (2000), in Remington: The Science and Practice of Pharmacy, Gennaro, A. R., ed., Lippincott Williams & Wilkins, Baltimore, pp. 808, 809.
Hugbo, P. G., Mendie, U. E., and Nasipuri, R. N. (1998), Int. J. Pharm. 167, 1–6.
Vessoni Penna, T. C., Marques, M., Machoshvili, I. A., and Ishii, M. (2002), Appl. Biochem. Biotechnol. 98–100, 539–551.
Avis, K. E. and Levchuck, J. W. (2000) in Remington: The Science and Practice of Pharmacy, Gennaro A. R., ed., Lippincott Williams & Wilkins, Baltimore, pp. 808–809.
United States Pharmacopeia, 24th ed. (2000), United States Pharmacopeial Convention, Rockville, MD, USA.
Errampali, D., Leung, K., Cassidy, M. B., Kostrzynska, M., Blears, M., Lee, H., and Trevors, J. T. (1999), J. Microbiol. Methods 35, 187–189.
Vessoni Penna, T. C., Ishii, M., Cholewa, O., and De Souza, L. C. (2004), Lett. Appl. Microbiol. 38, 135–139.
Ward, W. W. (1998), in Green Fluorescent Protein, Properties, Applications and Protocols, Chalfie, M. and Kain, S. (eds.), Wiley-Liss, New York, pp. 45–75.
Baier, S. and McClements, D. J. (2001), J. Agric. Food Chem. 49, 2600–2608.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Vessoni Penna, T. C. and Ishii, M. (2002), BMC Biotechnology, http://www.biomedcentral.com/1472-6750/2/7/qc.
Vessoni Penna, T. C., Ishii, M., Pessoa, A. Jr., Nascimento, L. O., Souza, L. C., and Cholewa, O. (2004), Appl. Biochem. Biotechnol. 113–116, 453–468.
Lide, D. R., ed. (1996), Handbook of Chemistry and Physics, 77th ed., CRC Press, Boca Raton, FL.
Vessoni Penna, T. C., Chiarini, E., Machoshvili, I. A., Ishii, M., and Pessoa, A. Jr., (2002), Appl. Biochem. Biotechnol. 98–100, 791–802.
Chiarini, E. and Vessoni Penna, T. C. (2003), Braz. J. Pharm. Sci. 39, 457–466.
McClements, D. J. (2001), Food Hydrocolloids 15, 355–363.
Marante, T. R., Ishii, M., Vessoni Penna, T. C., and Pessoa, A. Jr., (2003), Braz. J. Pharm. Sci. 39(3), 220–222.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Penna, T.C.V., Ishii, M., Kunimura, J.S. et al. Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values. Appl Biochem Biotechnol 122, 501–527 (2005). https://doi.org/10.1385/ABAB:122:1-3:0501
Issue Date:
DOI: https://doi.org/10.1385/ABAB:122:1-3:0501