Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium
| Autor: | Hidetsugu Asada, Mitsunori Shiroishi, Keisuke Ito, So Iwata, Tatsuro Shimamura, Takeshi Murata, Kiyohiko Igarashi, Takuya Kobayashi, Norimichi Nomura, Taishi Sugawara, Takami Yurugi-Kobayashi, Natsuko Tokuda |
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| Rok vydání: | 2011 |
| Předmět: |
DNA
Complementary Anion exchanger Size-exclusion chromatography Molecular Sequence Data Phanerochaete Antiporters Pichia Pichia pastoris Cell membrane Fungal Proteins Anion Exchange Protein 1 Erythrocyte medicine Humans Amino Acid Sequence Cloning Molecular Gene Purification Chrysosporium Phanerochaete chrysosporium Chromatography biology Cell Membrane biology.organism_classification Up-Regulation medicine.anatomical_structure Membrane Membrane protein Biochemistry Solubility Electrophoresis Polyacrylamide Gel Sequence Alignment Biotechnology Cloning |
| Zdroj: | Protein expression and purification. 79(1) |
| ISSN: | 1096-0279 |
| Popis: | Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl− and HCO 3 - 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. |
| Databáze: | OpenAIRE |
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