基础医学与临床 ›› 2013, Vol. 33 ›› Issue (1): 77-81.

• 研究论文 • 上一篇    下一篇

人睾丸组织TRIM69蛋白具有催化多聚泛素链形成的活性

韩永卿,李容,高晋兰,缪时英,王琳芳   

  1. 中国医学科学院 基础医学研究所
  • 收稿日期:2012-11-05 修回日期:2012-11-20 出版日期:2013-01-05 发布日期:2012-12-25
  • 通讯作者: 王琳芳 E-mail:wang.linfang@imicams.ac.cn
  • 基金资助:
    国家重大研究计划项目

The human testis TRIM69 protein possesses an activity of catalyzing formation of muti-ubiquitination

  • Received:2012-11-05 Revised:2012-11-20 Online:2013-01-05 Published:2012-12-25

摘要: 目的 验证人源TRIM69蛋白是否具有催化多聚泛素链形成的活性。方法 把人源TRIM69基因构建到原核细胞表达载体中,经过转化、诱导、表达和纯化获得TRIM69纯化蛋白,通过细胞外泛素化实验分析其是否具有催化多聚泛素链形成的活性;另外构建了TRIM69基因的全长、RING结构域的点突变、以及RING结构域截短序列的真核细胞表达载体,瞬时转染293T细胞或稳定表达泛素的HeLa细胞株,通过免疫沉淀结合免疫印迹方法检测其是否具有催化多聚泛素链形成的活性。结果 泛素化分析实验表明TRIM69具有催化多聚泛素链形成的活性并且其活性依赖于它的RING结构域。结论 成功鉴定了人源TRIM69蛋白具有催化多聚泛素链形成的活性,为进一步证明其是一个新的E3泛素连接酶奠定基础。

关键词: 精子发生, E3 泛素连接酶, TRIM, RING finger

Abstract: Objective To identify whether human TRIM69 could catalyze formation of multiubiquitinylated products. Methods The gene of human TRIM69 was cloned into a procaryotic expression vector, which was then transformed into E.coli. Purified protein of TRIM69 was obtained through the process of induction, expression, and purification, which was used in the following experiment of ‘in vitro ubiquitination assay’ to detect whether TRIM69 could catalyze formation of multiubiquitinylated products. In addition, we constructed TRIM69 gene full length or a point mutagenesis of RING domain sequence or a deleted RING domain sequence into a eukaryotic expression vector. Then the corresponding vectors were transiently transfected into HEK293T cells or HeLa cells of stably expressed ubiquitination. The experiment of immunoprecipitation combined with immunoblotting was performed to detect whether TRIM69 could catalyze formation of multiubiquitinylated products in cells. Results The ubiquitination assay results demonstrated that TRIM69 could catalyze formation of multiubiquitinylated products, which was dependent on its RING domain. Conclusion The enzyme activity of TRIM69 in catalyzing the formation of muti-uiquitinylated products was successfully identified, which builds up a basis for further characterizing TRIM69 as a novel E3 ubiquitin ligase.

Key words: spermatogenesis, E3 ubiquitin ligase, TRIM, RING finger

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