Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells
| Autor: | Nathan D. Pfeifer, Muhammed F. Hashim, William R. Tschantz, Ajith V. Kamath, Anthony J. Lanzetti, Caryl L. Meade, Françoise Berlioz-Seux, Leyu Wang |
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| Rok vydání: | 2007 |
| Předmět: |
Glycosylation
Glycoside Hydrolases Estrone Detergents Molecular Sequence Data Organic Anion Transporters Sf9 Biology Spodoptera Cell Line chemistry.chemical_compound Western blot medicine Animals Humans Amino Acid Sequence medicine.diagnostic_test Cell Membrane Molecular biology In vitro Recombinant Proteins Transport protein Kinetics Membrane chemistry Biochemistry biology.protein Heterologous expression Biotechnology Organic anion |
| Zdroj: | Protein expression and purification. 57(2) |
| ISSN: | 1046-5928 |
| Popis: | 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 μM 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 1.1 mg 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. |
| Databáze: | OpenAIRE |
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