重组人DFF45能抑制爪蟾卵非细胞体系中的凋亡特异核酸酶  

Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts

作　　者：杨巍[1] 卢智刚[1] 张岚[1] 翟中和[1] 

机构地区：[1]北京大学生命科学学院,北京100871

出　　处：《生物化学与生物物理学报》2001年第1期82-86,共5页

基　　金：国家重点基础研究发展规划项目!No .G19990 5 390 5&&

摘　　要：利用RT PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了人DNA断裂因子 (DFF) 4 5的cDNA ,将其克隆到pET 2 8a(+ )表达载体中 ,经IPTG诱导在大肠杆菌BL2 1(DE3)中获得了高效表达。表达的DFF45重组蛋白占菌体总蛋白质的 5 6 .6 % ,并位于细胞的可溶性组分中。利用融合在DFF45N端的His·Tag ,采用亲和层析的方法纯化重组蛋白 ,再经热变性处理去除杂蛋白后 ,获得了电泳纯的DFF45。将纯化的重组人DFF45加入到爪蟾卵非细胞凋亡体系中 ,可有效地抑制外源λDNA的水解 ,并可抑制外源鸡红细胞核形成凋亡特有的DNA梯。实验结果说明爪蟾卵非细胞体系中确实存在凋亡特异核酸酶 。The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD and 45 kD subunits. The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the genomic DNA degradation into nucleosomal fragments during apoptosis. As an inhibitor for DFF40, the 45 kD subunit (DFF45) complexes with DFF40, inhibiting DNase activity until certain apoptosis signals are received. In cells undergoing apoptosis, the cleavage of DFF45 by activated caspase 3 frees DFF40 from the complex and initiates the apoptosis specific DNA fragmentation. In this report, the coding region of human DFF 45 gene was amplified from the total RNA of HeLa cells by RT PCR. The resulting 1 kb DNA fragment was cloned into the bacterial expression vector pET 28a(+) with a 6×histidine tag fused to the N terminus of DFF45, generating plasmid pET28a DFF 45, which was then used to transform E.coli BL21(DE3). Induced by IPTG, the recombinant DFF45 was expressed efficiently with a yield of 56.6% of total bacterial proteins. The product was purified to homogeneity through a nickel affinity column, followed by heat treatment, and approximately 4—6 mg of DFF was purified from 100 ml culture. Purified recombinant human DFF45, added into the apoptotic cell free system of Xenopus egg extracts, could effectively inhibit both the digestion of λDNA and the degradation of chromosomal DNA into nucleosomal fragments in the nuclei of chicken red blood cells. Our results demonstrated the existence of an apoptosis specific endonuclease in this cell free system, the activity of which could be inhibited by recombinant human DFF45.

关 键 词：细胞凋亡 DNA裂因子(DFF)45 非细胞体系 细胞色素C 凋亡特异核酸酶 

分 类 号：Q25[生物学—细胞生物学]