Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.
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.
Tschantz
William R
WR
Department of Protein Engineering, Pfizer Inc., Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA. william.r.tschantz@pfizer.com
Pfeifer
Nathan D
ND
Meade
Caryl Lane
CL
Wang
Leyu
L
Lanzetti
Anthony
A
Kamath
Ajith V
AV
Berlioz-Seux
Francoise
F
Hashim
Muhammed F
MF
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
D003902:Detergents; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; C450859:SLCO2B1 protein, human; D004970:Estrone; D006026:Glycoside Hydrolases; C017296:estrone sulfate