| 王 颖,邹世清,陈 波.转录因子E2F1蛋白纯化及甲基化鉴定[J].现代生物医学进展英文版,2017,17(21):4012-4015. |
| 转录因子E2F1蛋白纯化及甲基化鉴定 |
| Purification and Methylation Identification of Transcription Factor E2F1 Protein |
| Received:December 27, 2016 Revised:January 22, 2017 |
| DOI：10.13241/j.cnki.pmb.2017.21.003 |
| 中文关键词: 原核表达 组蛋白甲基化转移酶 E2F1 SET7/9 |
| 英文关键词: Prokaryotic expression Histone methylation transferase E2F1 SET7/9 |
| 基金项目: |
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| 中文摘要: |
| 摘要 目的：构建人E2F1基因原核表达质粒pGEX-KG-E2F1，并在大肠杆菌中诱导表达 。随后验证纯化得到的E2F1蛋白可作为底物被甲基化转移酶修饰。方法：构建原核表达质粒pGEX-KG-E2F1，在大肠杆菌BL-21中经异丙基硫代半乳糖苷（IPTG）诱导表达，利用GST亲和层析法纯化表达的E2F1蛋白。随后将纯化的E2F1蛋白作为底物，组蛋白甲基化转移酶SET7/9作为酶进行体外同位素标记放射自显影实验，检测纯化的E2F1蛋白能否被甲基化。结果：酶切鉴定和测序结果证明成功构建了原核表达载体pGEX-KG-E2F1，SDS-PAGE检测结果证明实现了人E2F1基因在大肠杆菌中的可溶性表达，放射自显影证明纯化得到的E2F1蛋白可作为底物被甲基化转移酶SET7/9甲基化。结论：成功构建了转录因子E2F1体外甲基化体系,为筛选新的能甲基化E2F1的酶奠定基础。 |
| 英文摘要: |
| ABSTRACT Objective: To construct prokaryotic expression vector pGEX-KG-E2F1 of the human gene E2F1, and to express fusion protein and purify the E2F1 protein, and to validate that our purified E2F1 can be used as the substrate for histone methyltransferase-me- diated methylation. Methods: The prokaryotic expression plasmid pGEX-KG-E2F1 was constructed and the expression of E2F1 was in- duced by IPTG in E.coli BL-21. And then we use GST affinity chromatography to purify E2F1 protein. Then we use the purified E2F1 protein as a substrate, and the histone methyltransferase enzyme SET7/9 as the enzyme for autoradiography experiments in vitro. Results: Enzyme digestion and sequencing results demonstrated the prokaryotic expression vector pGEX-KG-E2F1 was constructed successfully. SDS-PAGE results proved the E2F1 protein can be solublely expressed and autoradiography demonstrated that purified E2F1 protein can be methylated by histone methylation transferase SET7/9. Conclusion: We successfully constructed E2F1 methylation system in vitro, and this work certainly laid a solid foundation for the further study of screening histone methyltransferases which can methylate E2F1. |
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