SARWAT, SUNEHERA
(2016)
Trials and tribulations in the expression and purification of ABCG2.
MRes thesis, University of Nottingham.
Abstract
ABC (ATP binding cassette) transporters are a diverse superfamily of membrane proteins, which couple the transport of substrate with the hydrolysis of ATP. ABCG2 (ATP binding cassette G2, also known as Breast Cancer Resistance Protein (BCRP), is a well-known multidrug efflux pump that transports a wide range of structurally unrelated chemotherapeutic drugs, such as mitoxantrone, methotrexate, topotecans, flavopiridol and tyrosine kinase inhibitors to name a few. Physiologically, ABCG2 is expressed and localized in different tissues of the body such as the placenta, gut and blood-brain-barrier where it plays a protective role and provides a first line of defence against environmental toxins. A single nucleotide polymorphism (SNP) in ABCG2 has been shown to be a significant causative aspect in the development of gout due to impaired urate transport.
Although several studies have been performed on this membrane protein, data regarding its structure is still missing. Thus, the main aim of this study was to bridge this knowledge gap by taking the first step and developing a robust purification method for heterologously expressed ABCG2. Initially, His6-tagged ABCG2 was overexpressed in Sf9 insect cells, and solubilised using a novel detergent free nanodisc-forming polymer styrene maleic acid (SMA). Although, solubilisation was remarkably effective, with the majority of ABCG2 being solubilised, purification using Ni-NTA resins was unsuccessful, potentially due to occlusion of the His tag. Thus, ABCG2 constructs expressing Strep and Strep-His13-tag were engineered in order to offer alternative affinity chromatography possibilities. Both constructs were well expressed in Sf9 cell membranes and again could be easily solubilized with SMA. However, purification using either metal affinity chromatography or streptactin affinity chromatography again gave the same negative result. Finally, a classical detergent based protein extraction using a mild detergent (dodecyl-β-D-maltopyranoside) along with E. coli total lipid extract was employed. This resulted in partial solubilisation of ABCG2, but notably the ABCG2 could bind to nickel resins and be enriched, laying the groundwork for optimization and further purification
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