sap18和sufu蛋白质复合物的表达纯化研究 - 中国细胞生物学学报.pdf

网络出版时间：2014-01-15 14:03 网络出版地址：/kcms/doi/10.11844/cjcb.2014.02.0296.html 中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(2): DOI: 10.11844/cj cb.2014.02.0296 SAP18和Sufu蛋白质复合物的表达纯化研究 安 影 吴 更* (上海交通大学生命科学技术学院, 上海200240) 摘要 通过分子克隆技术将Sufu和SAP 18构建到原核载体, 在原核表达系统进行外源表达, 采用亲和层析和分子筛层析对Sufu和SAP 18及其复合物进行纯化。同时利用非变性胶的方法进一 步确定该蛋白之间的相互作用及结合比例。结果表明, 原核表达系统中Sufu和SAP 18蛋白表达量高, 可以纯化到高纯度的蛋白质。Sufu和SAP 18结合的摩尔比为1:1, 按摩尔比1:2混合、纯化可以得到 高纯度、稳定的蛋白复合物, 从而为进一步复合物的结构生物学研究奠定基础。 关键词 SAP 18; Sufu; 表达; 纯化; 相互作用 Expression and Purification of Suppressor of Fused in Complex with Sin3-Associated Polypeptide of 18 kDa An Ying, Wu Geng* (School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China) Abstract Molecular cloning, prokaryotic expression and protein purification technology were used to ob- tain human SAP18, human Sufu and human SAP18-Sufu protein complex. The native PAGE method was used to identify the interaction and the ratio between SAP18 and Sufu proteins. The result demonstrated that SAP18 and Sufu proteins were highly expressed in the prokaryotic expression system. SAP18 interacts with Sufu by a molar ratio of 1:1. The high purity and stability of the SAP18-Sufu complex was obtained after the co-purification. These results laid a solid foundation for the future structural biology research on the protein complex. Key words SAP18; Sufu; expression; purification; interaction Hedgehog(Hh)信号通路在生物发育中起着重 法接近DNA而抑制其转录