Ono M, Tucker P W, Capra J D
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Nucleic Acids Res. 1994 Sep 25;22(19):3918-24. doi: 10.1093/nar/22.19.3918.
Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.
Ku是一种普遍存在的核异源二聚体蛋白，由p70和p86亚基组成，可结合双链DNA末端并在体内与染色体相关联。它最初被描述为某些自身免疫性疾病患者的自身抗原。Ku的各个亚基很难在不发生变性的情况下从人类细胞中分离出来，并且生产功能性重组Ku的尝试在很大程度上并不成功。在这里，我们利用两种重组杆状病毒载体，它们携带p70或p86 cDNA，并分别表达Ku亚基，以及将它们组装成完整的Ku异源二聚体。在电泳迁移率变动分析中，重组Ku与线性双链DNA结合，但不与超螺旋、带切口的环状或线性单链DNA结合。两个亚基自身都不结合DNA，这表明异源二聚化对于功能至关重要。我们还描述了一种使用六组氨酸标签分离高度纯化的重组Ku的简单纯化方法。杆状病毒表达系统不仅为p70和p86亚基，也为功能性Ku异源二聚体提供了稳定而有效的来源。
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