Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

Abstract

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

“…Cloning of Human MDR3 and Site-directed MutagenesisWe cloned human wild type MDR3 (NM_000443.3) as described previously (18). Site-directed mutagenesis was carried out with the QuikChange XL (Agilent Technologies) and the Phusion site-directed mutagenesis kit (Thermo Scientific), respectively.…”

“…Site-directed mutagenesis was carried out with the QuikChange XL (Agilent Technologies) and the Phusion site-directed mutagenesis kit (Thermo Scientific), respectively. To generate the ATPase-deficient mutant, we exchanged Glu-558 and Glu-1207 of the conserved Walker B motif against Gln with the primer pair as described before (18). The Q1174E mutant was introduced into MDR3 with the Phusion site-directed mutagenesis kit (Thermo Scientific) using forward primer 5Ј-GATAAGGGGACTGAGCTCTCAGGA-GGTCAAAAAC-3Ј and the reverse primer 5Ј-CCCACTCTT-GTTTCATATTTGTGGGGTAACG-3Ј.…”

“…Transformation of P. pastoris and Expression Screening-MDR3 expression constructs were transformed into electrocompetent P. pastoris X33 cells (Invitrogen) using standard procedures, and the expression level was analyzed as described in Ellinger et al (18).…”

“…Solubilization and Purification of MDR3-The purification of wild type MDR3 or mutant was performed as described previously with a few modifications (18). All procedures were carried out at 4°C.…”

“…Previously, we demonstrated that human wild type MDR3 exhibited PC-induced ATPase activity, whereas the ATPasedeficient mutant of both Walker B motifs did not show stimulation (18). In this study, we characterized the ATPase activity of wild type MDR3 in terms of kinetic parameters, substrate spectrum, and the effect of phosphate analogues.…”