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| Intein-mediated Soluble Expression, Purification and Functional Identification of Human AR DBD in E. coli |
| YAO Guang-xin1, ZHANG Yi-xuan1, HU Shuang-gang2,3 |
1. Shenyang Pharmaceutical University, Shenyang 110016, China;
2. Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Institute of Biochemistry and Cell Biology, Shanghai 200031, China;
3. State Key Laboratory of Molecular Biology, Shanghai 200031, China |
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Abstract Androgen receptor (AR) is a transcription factor belonging to Nuclear Hormone Receptor Superfamily and its DNA binding domain (DBD) is essential for its binding affinity and specificity to androgen response element. Traditional method to obtain the recombinant AR DBD is fusing it to glutathione S epoxide transferase or Staphylococcus aureus proteinA in order to improve the solubility and yield. The question is the fusion tag might affect the characterization of target protein, and removing of the fusion tag is usually cumbersome. In order to obtain functional AR DBD without fusion tag in an easy and convenient way, the sequence encoded human AR 520~644 amino acids which includes the DBD is amplified by PCR, and then inserted into pTWIN1 vector. After optimizing of the inducing temperature and IPTG concentration, almost all recombinant protein is expressed in a soluble manner at 20℃ and 30℃ in host E.coli strain BL21(DE3). The whole cell lysate is then loaded to Chitin beads and the optimal pH for self-cleavage is determined. Using this method the AR DBD without any fusion tags is purified by only one step and the product shows high purity and good performance in electrophoretic mobility shift assay.
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Received: 07 January 2011
Published: 26 April 2011
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