The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides
Date
2009
Authors
Heikal, Adam
Box, Karl
Rothnie, Alice
Storm, Janet
Allen, Marcus
Callaghan, Richard
Journal Title
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Volume Title
Publisher
Academic Press
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.
Description
Keywords
Keywords: adenosine triphosphatase; glycerol; maltose; multidrug resistance protein 1; trehalose; animal cell; article; controlled study; enzyme activity; freeze drying; high throughput screening; nonhuman; priority journal; protein purification; protein stability; ABCB1; ATPase; Chemotherapy; High throughput; Liposome; Lyophilisation; Multidrug resistance; Proteoliposome; Purification; Trehalose
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Source
Cryobiology
Type
Journal article
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2037-12-31