title: Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.
abstract: ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.
authors.forename: Naomi L
authors.initials: NL
authors.lastname: Pollock
authors.forename: Christopher A
authors.initials: CA
authors.lastname: McDevitt
authors.forename: Richard
authors.initials: R
authors.lastname: Collins
authors.forename: Petronella H M
authors.initials: PH
authors.lastname: Niesten
authors.forename: Stephen
authors.initials: S
authors.lastname: Prince
authors.forename: Ian D
authors.initials: ID
authors.lastname: Kerr
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
authors.forename: Richard
authors.initials: R
authors.lastname: Callaghan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D003124:Colorimetry; D015870:Gene Expression; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry*; D008854:Microscopy, Electron; D008958:Models, Molecular; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells / Q000821:virology*; D013696:Temperature
chemical_list: C056475:ABCA4 protein, human; C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D008563:Membrane Lipids; D011994:Recombinant Proteins
keywords: ABCA4; ATP Binding Cassette transporter; Lipid–protein interaction; Membrane protein structure; Membrane transport; P-glycoprotein