title: The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides.
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 degrees C. For example, at 20 degrees 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.
authors.affiliation: University of Brighton, School of Pharmacy and Biomolecular Sciences, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK.
authors.forename: Adam
authors.initials: A
authors.lastname: Heikal
authors.affiliation: Sirius Analytical Instruments Ltd. Riverside, Unit 12 Forest Row Business Park Forest Row, East Sussex RH18 5DW, UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Box
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences Level 4, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Rothnie
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences Level 4, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, UK.
authors.forename: Janet
authors.initials: J
authors.lastname: Storm
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences Level 4, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Callaghan
authors.affiliation: University of Brighton, School of Pharmacy and Biomolecular Sciences, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK. Electronic address: m.c.allen@brighton.ac.uk.
authors.forename: Marcus
authors.initials: M
authors.lastname: Allen
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D004187:Disaccharides; D005612:Freeze Drying / Q000379:methods*; D005990:Glycerol; D006801:Humans; D008320:Maltose; D009036:Moths / Q000235:genetics; D044367:Phase Transition; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014199:Trehalose
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004187:Disaccharides; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D008320:Maltose; D014199:Trehalose; D000251:Adenosine Triphosphatases; D005990:Glycerol