title: Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.
abstract: OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.
authors.affiliation: Department of Protein Engineering, Pfizer Inc., Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA. william.r.tschantz@pfizer.com
authors.forename: William R
authors.initials: WR
authors.lastname: Tschantz
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Pfeifer
authors.forename: Caryl Lane
authors.initials: CL
authors.lastname: Meade
authors.forename: Leyu
authors.initials: L
authors.lastname: Wang
authors.forename: Anthony
authors.initials: A
authors.lastname: Lanzetti
authors.forename: Ajith V
authors.initials: AV
authors.lastname: Kamath
authors.forename: Francoise
authors.initials: F
authors.lastname: Berlioz-Seux
authors.forename: Muhammed F
authors.initials: MF
authors.lastname: Hashim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
chemical_list: D003902:Detergents; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; C450859:SLCO2B1 protein, human; D004970:Estrone; D006026:Glycoside Hydrolases; C017296:estrone sulfate