[FOXO1 DNA结合结构域的表达、纯化及其DNA特性]

[Expression and purification of FOXO1 DNA binding domain and its DNA properties].

作者信息

Ha Yinuer, Li Jun, Chen Yongheng, Chen Lin, Chen Zhuchu

机构信息

Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2017 Jan 28;42(1):1-7. doi: 10.11817/j.issn.1672-7347.2017.01.001.

DOI:10.11817/j.issn.1672-7347.2017.01.001

PMID:28216490

Abstract

To express and purify FOXO1 DNA binding domain (DBD) and to evaluate its DNA binding properties.  Methods: Gene sequence of FOXO1-DBD was optimized and the recombinant strains were induced at low temperature to obtain soluble protein of FOXO1-DBD. Then, FOXO1-DBD protein was purified through Ni affinity chromatography and cation exchange. Finally, the DNA binding properties of FOXO1-DBD was evaluated by electrophoretic mobility shift assay (EMSA).  Results: Most of soluble proteins were obtained by optimizing its genes sequence and induction at 21 ℃. Over 95% purity of FOXO1-DBD protein was obtained through two-steps purification. Purified protein exhibited good binding property to the DNA binding motif (G/ATAAACA) of FOX family.  Conclusion: An efficient expression and purification method for FOXO1-DBD is established, and the complexity of FOXO1 protein in recognition of DNA is verified.

摘要

表达并纯化叉头框蛋白O1（FOXO1）的DNA结合结构域（DBD），并评估其DNA结合特性。方法：优化FOXO1-DBD的基因序列，低温诱导重组菌株以获得FOXO1-DBD的可溶性蛋白。然后，通过镍亲和层析和阳离子交换纯化FOXO1-DBD蛋白。最后，采用电泳迁移率变动分析（EMSA）评估FOXO1-DBD的DNA结合特性。结果：通过优化基因序列并在21℃诱导，获得了大部分可溶性蛋白。经两步纯化，FOXO1-DBD蛋白的纯度超过95%。纯化后的蛋白对FOX家族的DNA结合基序（G/ATAAACA）表现出良好的结合特性。结论：建立了一种高效表达和纯化FOXO1-DBD的方法，并验证了FOXO1蛋白识别DNA的复杂性。