title: Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium.
abstract: Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl(-) and HCO3(-)across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-β-D-maltoside. His-tagged PcAEP was purified as a single band on SDS-PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins.
authors.affiliation: Department of Medical Chemistry, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tokuda
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Igarashi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics; D017920:Antiporters / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D002462:Cell Membrane / Q000648:ultrastructure; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D016415:Sequence Alignment; D012995:Solubility; D015854:Up-Regulation
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D018076:DNA, Complementary; D005656:Fungal Proteins