TY - JOUR
T1 - Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium
AU - Tokuda, Natsuko
AU - Igarashi, Kiyohiko
AU - Shimamura, Tatsuro
AU - Yurugi-Kobayashi, Takami
AU - Shiroishi, Mitsunori
AU - Ito, Keisuke
AU - Sugawara, Taishi
AU - Asada, Hidetsugu
AU - Murata, Takeshi
AU - Nomura, Norimichi
AU - Iwata, So
AU - Kobayashi, Takuya
N1 - Funding Information:
We thank Ms. S. Funamoto, Ms. N. Katsuta, Ms. T. Uemura and Dr. Y. Nomura for technical assistance, and Ms. C. Fukui for secretarial assistance. This work was supported by a Research Grant from the ERATO Iwata Human Receptor Crystallography Project from the Japan Science and Technology Agency (JST) (to S.I.), in part by a Grant-in-Aid for Scientific Research (B) ( 20370035 to T.K. and 21370043 to T.S.), by a grant from Takeda Science Foundation (to T.K.), by a grant form the Ichiro Kanehara Foundation (to T.K. and T.S.), by a grant from The Sumitomo Foundation (to T.K.), by a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.K.), by a Research Fellowship from the Uehara Medical Foundation (to T.K.), and by a Challenging Exploratory Research ( 22659059 to T.K.), and in part by a Research Grant from the Membrane Protein Structure Initiative (MPSi) (to S.I.).
Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2011/9
Y1 - 2011/9
N2 - 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.
AB - 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.
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U2 - 10.1016/j.pep.2011.04.006
DO - 10.1016/j.pep.2011.04.006
M3 - Article
C2 - 21515379
AN - SCOPUS:79960137294
VL - 79
SP - 81
EP - 87
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -