title: Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.
abstract: Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies.
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
authors.forename: Shahid
authors.initials: S
authors.lastname: Rehan
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland. Electronic address: veli-pekka.jaakola@oulu.fi.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D061987:Sf9 Cells; D013552:Swine
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D011994:Recombinant Proteins; C438649:SLC29A1 protein, human
keywords: Anti-cancer; Equilibrative nucleoside transporter; Expression; Purification; Thermostability