title: Analysis of recombinant tagged equilibrative nucleoside transporter 1 (ENT1) expressed in E. coli.
abstract: Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ~15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.
authors: Department of Biology, York University, Toronto, ON, Canada.
authors: German
authors: G
authors: Reyes
authors: Nicole M I
authors: NM
authors: Nivillac
authors: Maria
authors: M
authors: Chalsev
authors: Imogen R
authors: IR
authors: Coe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D011994:Recombinant Proteins