Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium

Natsuko Tokuda, Kiyohiko Igarashi, Tatsuro Shimamura, Takami Yurugi-Kobayashi, Mitsunori Shiroishi, Keisuke Ito, Taishi Sugawara, Hidetsugu Asada, Takeshi Murata, Norimichi Nomura, So Iwata, Takuya Kobayashi

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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.

Original languageEnglish
Pages (from-to)81-87
Number of pages7
JournalProtein Expression and Purification
Volume79
Issue number1
DOIs
Publication statusPublished - Sep 1 2011

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All Science Journal Classification (ASJC) codes

  • Biotechnology

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