癌蛋白PRC17与人肌球蛋白调节轻链相互作用的初步鉴定-东南大学学报医学版

癌蛋白PRC17与人肌球蛋白调节轻链相互作用的初步鉴定

单位：1. 东南大学医学院病理学与病理生理学系, 江苏南京 210009;
2. 东南大学生命科学研究院, 江苏南京 210009

分类号：Q78

出版年·卷·期（页码）：2014·33·第一期（12-17）

摘要：

目的：分析前列腺癌基因17（PRC17）与人肌球蛋白调节轻链（MLC2）之间的相互作用，并鉴定PRC17蛋白序列上与MLC2相互作用的功能区域。方法：经大肠杆菌表达并用Glutathione Sepharose 4B纯化GST及融合蛋白GST-MLC2，用SMMC-7721细胞表达带HA标签的PRC17及其截短突变体HA-PRC17（353）、HA-PRC17（251）、HA-PRC17（164）和HA-PRC17（Δ164），GST 沉淀实验分析GST-MLC2与PRC17及其截短体的相互作用。结果：经诱导表达并纯化的GST和GST-MLC2分子质量符合预期，分别约为28.4 kD和46.6 kD，纯度均95%以上；GST 沉淀实验结果显示，PRC17结合MLC2，不结合阴性对照GST；PRC17（353）和PRC17（251）也能有效结合MLC2，但PRC17（164）和PRC17（Δ164）都不能与MLC2结合。结论：PRC17能特异性结合MLC2，其蛋白序列上与MLC2结合的部位位于其氨基端251位氨基酸之内，可能在164位氨基酸附近。

Objective: To analyze the interaction between PRC17 and MLC2 and identify MLC2-binding site on PRC17. Methods: GST (glutathione-s-transferase) and GST-MLC2 fusion protein were expressed in E. coli BL21 and purified by Glutathione Sepharose 4B affinity chromatography. HA-tagged PRC17 and truncated PRC17 mutants HA-PRC17(353), HA-PRC17(251), HA-PRC17(164)and HA-PRC17(Δ164)were expressed in SMMC-7721 cells. The GST-pull down assay was performed to test the interaction of MLC2 with HA-PRC17 and PRC17 mutants respectively. Results: GST and GST-MLC2 were induced to be expressed in E.coli BL21, and their molecular weights were consistent to the anticipated molecular weights,28.4 kD and 46.6 kD respectively.The purity of both GST and GST-MLC2 was above 95% respectively;GST pull-down assay showed that PRC17 could bind to GST-MLC2, but could not bind to negative control GST protein;PRC17(353)and PRC17(251)could bind to MLC2 as well, however, PRC17(164)and PRC17(Δ164)could not bind to MLC2. Conclusion: PRC17 could interact specifically with MLC2 within 251 aminos in N-terminal of PRC17,in particular around the 164th animo.

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