The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

Heikal, Adam, Box, Karl, Rothnie, Alice, Storm, Janet, Callaghan, Richard and Allen, Marcus (2009). The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides. Cryobiology, 58 (1), pp. 37-44.

Abstract

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

Publication DOI: https://doi.org/10.1016/j.cryobiol.2008.10.125
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences
Additional Information: © 2009, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/
Uncontrolled Keywords: adenosine triphosphatases,animals,differential scanning calorimetry,disaccharides,freeze drying,glycerol,humans,maltose,moths,p-glycoprotein,phase transitions,proteolipids,recombinant proteins,temperature,trehalose,ABCB1,multidrug resistance,lyophilisation,high throughput,liposome,ATPase,proteoliposomes,chemotherapy,purification,Agricultural and Biological Sciences(all),Biochemistry, Genetics and Molecular Biology(all),Medicine(all)
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2009-02
Authors: Heikal, Adam
Box, Karl
Rothnie, Alice ( 0000-0002-4259-7015)
Storm, Janet
Callaghan, Richard
Allen, Marcus

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